Chip Technology

Preface Preface DNA-chip analysis has come a long way since the first official gathering of scientists working in this...

Author: Jorg D Hoheisel

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Preface

Preface

DNA-chip analysis has come a long way since the first official gathering of scientists working in this field, which took place in Moscow in 1991, comprising about 50 scientists from Europe and the USA. Then, the initial aim was the development of a technology for high-throughput sequencing of the human genome, named sequencing by hybridisation. The field soon diversified, however, in terms of methodology and application. Nowadays, DNA-microarrays seem to be a common commodity in biological sciences. The complexity hidden behind the apparent ease of such studies, however, is highlighted by the fact that it took about ten years of technology development – and persuasion – before the methodology really took off.Also, at closer scrutiny one realises that many problems still need to be resolved and only relatively limited inquiries have been attempted so far considering the potentially possible. Nevertheless, even these experiments have produced data on a scale beyond imagination for most people in biology a few years ago and for many even today. Even the data sets originating from large-scale sequencing are dwarfed by the quantity of information from chip-based analyses. Thus, DNA-microarray technology will be the true herald of change in biology. Having developed earlier from a descriptive to an analytical science, biology will split in future into an experimental and a theoretical branch, similar to what happened in physics quite a long time ago. This change in biology is no better represented than by the authors of this book, who took part in bringing about this shift in emphasis. They are wellknown experts in the field, many – like Edwin Southern, Hans Lehrach, Radoje Drmanac, Pavel Pevzner and Charles Cantor – have been actively pursuing array technology for more than a decade. Rather than pondering on the good old times, however, they demonstrate the continuous development in both technology and application areas and elucidate some critical points that need to be considered, when performing microarray analyses. The first article, by Pavel Pevzner and co-workers, informs on aspects of designing DNA-arrays, which is not a trivial undertaking, although rarely discussed. Even at this level of array-based analysis – right at the start rather than the end – informatics is required in order to deal with the inherent complexity. Such a design phase is followed by the actual production process.Although by no means the sole procedure for doing so, photolithographically controlled in situ synthesis is currently the best advanced technique for large-scale chip production with a high degree of reproducibility. Glen McGall and Fred Christians report on the procedures involved, some future developments and applications.

X

Preface

Muhammad Sohail and Edwin Southern describe in their contribution a different process for the creation of oligonucleotide arrays. Subsequently, they used the arrays in the screening for effective antisense reagents. This study is fascinating because of its impact on the understanding of interactions between nucleic acids, an interesting research area even after 50 years of structural DNA analysis, and in view of its practical implications for applications in biotechnology and molecular medicine. Hubert Köster, Charles Cantor and co-workers combine two high-throughput techniques – arrays with detection by mass spectrometry – for genotyping analyses. With the existence of about 1.5 million publicly known single nucleotide polymorphisms (SNPs), the exploration of this resource is a major challenge in the effort of translating basic genomics into applied, medically relevant molecular genetics. The work of Radoje Drmanac extends the objective of genotyping analyses and, at the same time, returns to the origin of DNA-chip analysis by pursuing ‘sequencing by hybridisation’, which is nothing short of performing a SNP analysis for each and every nucleotide of a given DNA-fragment. He reports recent achievements and deliberates on the exciting opportunities of this methodology. The text of Holger Eickhoff and colleagues already reaches beyond the mere DNA-chip by reporting on procedures that extend strongly into the field of proteomics, thus linking the two areas. Only by such measures, carried out experimentally as well as in silico, the complexity of functions in cellular systems will eventually be unravelled. Considering how array analyses are performed, it is only natural that a contribution on bioinformatics tools and databases should come at the end of the list. Its position does not reflect its importance, however. As a matter of fact, all preparatory work going into the production of the nice looking, colourful pictures from DNA-arrays is a useless squander unless it is assessed and presented in a way that makes the data accessible to human interpretation. Currently, much of the data produced on microarrays is actually wasted. Transcriptional profiling studies, for example, usually concentrate on few, specific biological aspects and ignore much else contained in the very set of raw data. This information could be useful for other studies, if only one could access it. For the purpose of going back to results for entirely different analytical purposes, central databases with appropriately designed and standardised procedures, as well as a common ontology, are essential. Alvis Brazma and colleagues have been instrumental in getting such efforts started. Overall, the various articles provide a good mix, covering many, although not all, aspects of microarray-based analysis, the latter no longer achievable in a single book, for the days of the Moscow meeting in 1991 are long past and the breadth of the field has expanded enormously both in terms of its technical aspects and the variety of potential applications. Nevertheless, I hope the picture is comprehensive enough for understanding the basics, elaborate enough to inform in detail on certain aspects and speculative enough to stimulate further developments. Heidelberg, May 2002

Jörg D. Hoheisel

CHAPTER 1

Combinatorial Algorithms for Design of DNA Arrays Sridhar Hannenhalli 1 · Earl Hubbell 2 · Robert Lipshutz 2 · Pavel A. Pevzner 3 1

Departments of Mathematics, University of Southern California, Los Angeles, California 90089-1113, USA. Present address: Celera Genomics, Rockville, Maryland 20850, USA 2 Affymetrix, 3380 Central Expressway Santa Clara, California 95051, USA. E-mail: [email protected] 3 Departments of Computer Science and Engineering, University of California at San Diego, La Jolla, California 92093-0114, USA. E-mail: [email protected]

Optimal design of DNA arrays requires the development of algorithms with two-fold goals: reducing the effects caused by unintended illumination (border length minimization problem) and reducing the complexity of masks (mask decomposition problem).We describe algorithms that reduce the number of rectangles in mask decomposition by 20–30% as compared to a standard array design under the assumption that the arrangement of oligonucleotides on the array is fixed. This algorithm produces provably optimal solution for all studied real instances of array design. We also address the difficult problem of finding an arrangement which minimizes the border length and come up with a new idea of threading that significantly reduces the border length as compared to standard designs. Keywords: DNA arrays, Photolitography, Mask design, Combinatorial algorithms, Rectangle cover problem

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

2

Placement Problem

. . . . . . . . . . . . . . . . . . . . . . . . . .

3

3

DNA Arrays and the Traveling Salesman Problem . . . . . . . . . .

5

4

Threading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

5

Rectangle Cover Problem

6

Mask Decomposition Problem

7

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

8

References

. . . . . . . . . . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . . . . . . . 14

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Advances in Biochemical Engineering/ Biotechnology, Vol. 77 Managing Editor: T. Scheper © Springer-Verlag Berlin Heidelberg 2002

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S. Hannenhalli et al.

1 Introduction DNA arrays have been greatly successful in a variety of genomic analyses [2], ranging from detecting SNPs [13] to functional genomics [3, 9, 14]. One method of synthesizing an oligonucleotide array is by a photolithographic V LSIPS (very large scale immobilized polymer synthesis) method similar to that used in the semiconductor industry. In this method, light is used to direct synthesis of oligonucleotides in an array. In each step, light is selectively allowed through a mask to expose cells in the array, activating the oligonucleotides in that cell for further synthesis. For every synthesis step there is a mask with corresponding open (allowing light) and closed (blocking light) cells. Each mask corresponds to a step of combinatorial synthesis described in [5]. The proper regions are activated by illuminating the array through a series of masks. Unfortunately, because of diffraction, internal reflection, and scattering, points close to the border between an illuminated region and a shadowed region are often subject to unintended illumination [5]. In such a region, it is uncertain whether a nucleotide will be appended or not. This uncertainty gives rise to probes with unknown sequences complicating interpretation of the experimental data. Methods are being sought to minimize the lengths of these borders so that the level of uncertainty is reduced. The problem is important not only for Affymetrix arrays but also for any other in-situ synthesis scheme, such as the recently published micromirror arrays [12]. Border Minimization Problem (BMP) is to arrange the probes on the array in such a way that the overall border length of all masks is minimal. For two probes x and y, let d (x, y) be the Hamming distance between probes, i.e., the number of positions in which x and y differ.An intuitive approach to BMP is to position similar probes at neighboring sites. In a synchronous array design when every iteration generates a fixed nucleotide at i-th positions of the probes, the overall border length of all masks equals Â d (x, y), where the sum is taken over all pairs of neighboring probes on the array. For uniform arrays this observation establishes the connection between minimization of border length and Gray codes [11]. An l-bit Gray code is defined as a permutation of the binary numbers between 0 and 2l –1 such that neighboring numbers have exactly one differing bit, as do the first and last numbers. For a fixed set of n probes, consider an n-vertex complete graph with edge weights equal to Hamming distances between probes. For uniform arrays with 4l probes, every solution of the Traveling Salesman Problem (TSP) corresponds to a Gray code and leads to arrangements that position similar probes in neighboring sites thus minimizing the border length of the mask for uniform arrays [11]. However, for assay arrays with arbitrary probes used in functional genomics, the border length minimization remained an open proplem. The goal of positioning similar probes in neighboring sites led to the attempts of using TSP approach for border length minimization in the design of dedicated functional genomics arrays. In the very first design of such arrays at Affymetrix in mid 1990s Earl Hubbell implemented a greedy TSP algorithm for minimizing the border length of the assay chip. The motivation for using a TSP tour is that

Combinatorial Algorithms for Design of DNA Arrays

3

consecutive probes in the tour are likely to be similar. The probe arrangement corresponding to found TSP solution was further threaded on an array in a rowby-row fashion. The deficiency of this approach is that although probes in the same row are usually similar, the neighboring probes in the same column may be very different thus leading to an increase in border length. In this paper we propose a different idea for array design called optimal threading. Define a threading as a self-avoiding path through the sites of an array. Different threadings vary in the border length and we show that a clever choice of threading reduces the border length by 20% for large chips (as compared to the row-by-row threading). Masks for DNA arrays are built by patterning equipment which generates rectangular regions by a long series of “flashes”, each flash producing a rectangle. Complex masks are made by consecutive generation of rectangles. The cost of a mask is (approximately) linear in the number of rectangles generated, and there is a fixed upper limit to the number of rectangles it is feasible to generate, before a different (expensive) mask fabrication technology must be used instead. Therefore, we are interested in a cover of a mask with a minimum number of rectangles (the rectangles in a cover can overlap). The rectangle cover problem is known to be NP-hard and approximation algorithms with small performance ratios for this problem are unknown ([6, 7]). We explore the specifics of oligonucleotide masks and devise an efficient algorithm which finds a (provably) optimal rectangle cover for all masks we tested at Affymetrix. The paper is organized as follows. In the following section we present the “Placement problem” which is a generalization of a few interesting optimization problem, including optimal mask design and (surprisingly) protein folding in the lattice model. We further describe applications of TSP for DNA array design and present threading as novel approach to optimizing the border length. Finally, we describe the algorithms for optimal mask decomposition into rectangles.

2 Placement Problem Let G1 (V1 , E1 , w1) and G2 (V2 , E2 , w2) be two complete edge-weighted graphs with weight functions w1 and w2 .A bijective function y :V2 Æ V1 is called a placement of G2 on G1 . The cost of placement is defined as c(y)=

Â x, y Œ V2

w1 (x, y) w2 (y (x), y (y)).

The optimal placement problem is to find a placement of G2 on G1 of minimal cost. For optimal mask design problem the N¥N vertices of the grid graph G1 correspond to a N¥N array. The distance between grid cells (i1 , j1) and (i2 , j2) in the Manhattan metric is defined as |i2 –i1 |+| j2 –j1 |. The vertices of the grid are neighbors if the distance between them is 1. The weight w1 (x, y) is defined as 1 if x and y are neighbors and 0 otherwise. The vertices of the probe graph G2 correspond to N¥N probes to be synthesized on a chip. The weight function w2 (p, q) is de-

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S. Hannenhalli et al.

Table 1. Performance comparison of various algorithms for real masks

Chip Size

549a8 312¥312

549a14 312¥312

549a18 312¥312

549a19 312 ¥312

549a22 312 ¥312

#Black Cells #Maximal Rectangles #Prime Rectangles #Black Cells after Phase 1 #iterations in Phase 2 #Rectangles found in Phase 2 #Rectangles found after Phase 2 #Rectangles found by Rectangle_Cover #Rectangles found by Rectangle_Partition #Rectangles found by Rectangle_Greedy

17377 1394 1392 4

11152 993 968 260

14279 2362 2327 610

32519 2952 2920 509

16102 3425 3387 433

1

1

1

1

1

2

17

35

32

34

0

0

0

0

0

1394

985

2362

2952

3421

1394

985

2507

3406

3521

1513

1432

2980

4588

3816

fined as the distance between probes p and q. The distance between probes is the number of times an edge occurs between them in the entire set of masks to fabricate the chip. Depending on the synthesis strategy the distance is either Hamming distance or the distance defined by the synthesis-schedule. In such formulation the optimal placement attempts to position similar probes in the neighboring position of the grid and minimizes the overall border length of the masks. In many cases the overall border length is well-correlated with the number of flashes to produce the mask and therefore, the optimal placement reduces the cost of the mask set in these cases (Table 1). TSP is a particular case of the optimal placement problem (in this case G1 is a graph with edges of weight 1 forming a path and all other edges of weight 0). This observation motivates the following approach to optimal mask design problem: find TSP in the probe graph and thread it in a row-by-row fashion on the array grid. Such approach optimizes the placement for half of the edges (for adjacent cells in the same row) in the grid but completely ignores another half (for adjacent cells in the same column). Experiments suggest that this approach reduces the cost of the placement by 27% as compared to the random design for 64 Kb chip. The statistical analysis indicates that the larger is the chip the larger is the saving in cost (four-fold increase in the size of the chip increase the saving by about 5%). Our goal is to optimize the placement for the remaining half of the edges and to reduce the cost of the edges by additional 27%. Below we show how to reduce it by 20% thus achieving savings which is close to optimal.

5

Combinatorial Algorithms for Design of DNA Arrays

3 DNA Arrays and the Traveling Salesman Problem Since any improvement of the TSP-tour would likely improve the parameters of array design, we first tried to find a better TSP algorithm for assay arrays. Computing an optimal TSP tour for a graph with 64,000 vertices is not feasible. Note that only very fast approximation algorithms (i.e. at most quadratic) are suitable for our size of the problem. We implemented 2-opt algorithm [8] for TSP. Given an ordering of vertices p = p1 … pi–1 , pi , pi+1 … pj–1 , pj , pj+1 … pn , a reversal of p at i, j is a permutation p´= p1 … pi–1 , pj , pj–1 … pi+1 , pi , pj+1 … pn obtained from p by reversing the fragment pi , pi+1 … pj–1, pj . 2-opt neighborhood of p is formed by all permutations obtained from p by reversals. The Lin-Kernighan local improvement algorithm finds a suboptimal TSP tour by starting from an arbitrary permutation of vertices p = p (0). At a general step, given a permutation p (i), it searches for a permutation p (i+1) from 2-opt neighborhood of p (i) such that the cost of p (i +1) is less than the cost of p (i). The computation terminates at a local minimum when no such permutation in 2-opt neighborhood exists. Disappointingly, we found that a 2-opt optimization of the greedy TSP-tour leads to only 0.3% improvement (Table 1). We tried a few approaches to further improve the TSP solution (or to verify its proximity to the optimal solution), in particular, solving minimum length cycle cover problem with further merging of cycles in the cover into a hamiltonian path. A cycle cover is a set of (simple) cycles such that each vertex in the graph is contained in exactly one cycle. The Table 2. Performance comparison of various algorithms for random masks with varying density; the parameter p denotes that probability of a grid cell being a black cell

Chip Size

r(p=0.9) 500¥500

r(p=0.75) r(p=0.5) 500¥500 500¥500

r(p=0.25) 500 ¥500

r(p=0.1) 500¥500

#Black Cells #Maximal Rectangles #Prime Rectangles #Black Cells after Phase 1 #iterations in Phase 2 #Rectangles found in Phase 2 #Rectangles found after Phase 2 #Rectangles found by Rectangle_Cover #Rectangles found by Rectangle_Partition #Rectangles found by Rectangle_Greedy

224660 50944 8452 97522

186891 65399 28608 37771

123951 61364 48399 6400

60791 41092 39627 339

24241 20362 20322 24241

7

11

5

2

1

1343

9599

3513

272

1

12561

2393

18

0

0

22356

40600

51930

39899

20323

22726

43633

53123

39977

20323

28420

45900

55077

40267

20337

6

S. Hannenhalli et al.

weight of the cycle cover is the total length of its cycles. A minimum weight cycle cover can be reduced to matching problem in the bipartite graph. To solve the cycle cover problem we used Ed Rothberg’s implementation of Gabow’s weighted matching algorithm (available at dimacs.rutgers.edu/pub/netflow/ maxflow). However, for such large matching problems this software takes a lot of time even for 9Kb arrays and becomes prohibitively slow for larger arrays. The cost of the minimum length cycle cover provides a lower bound on the length of TSP-tour. Since cycle cover for large graphs is hard to compute we used the following lower bound that is just slightly worse than the cycle cover: 1 Â sum of costs of two shortest edges at vertex v, 22 v where the sum is taken over all vertices of the graph. For 9K chips this bound is just 0.4% worse than the bound based on the minimum length cycle cover problem. This bound demonstrates that the greedy algorithm is already within 5% of the optimal solution while the best algorithm we developed (Global Greedy) is within 3% of the optimal solution (Table 1).An explanation for such an excellent performance of the greedy algorithm is that the distances between probes in the assay arrays are “almost random” (compare with [1]). Global_Greedy (pseudocode shown below) algorithm proved to be the best in our tests of different approaches for TSP problems associated with DNA arrays. Input: Complete edge weighted graph G (V, E) Output: Path graph H (V, E´) Algorithm Global_Greedy 1. E´¨ F 2. while H (V, E´) is not connected 3. e ¨ the shortest edge in E 4. if H (V, E´» {e} has a cycle or a vertex of degree 3 5. delete edge e from E 6. else 7. E´¨ E´» {e} 8. Output H(V, E´)

4 Threading Since further improving of TSP tour is infeasible we turned to another idea. In this section we show how a choice of threading could significantly improve the border length for a given TSP tour over the set of probes. Figure 1 presents two different threadings of the same TSP tour. The neighbors on the grid that are not adjacent along the TSP tour are likely to have high cost.We refer to them as “bad” edges.At first glance it looks like threading does not affect the cost of placement. Figure 1a has nine “bad” (vertical) edges (shown by dashed and dotted lines). Figure 1b has nine other “bad” edges (shown by dashed and dotted lines). One can assume that the average cost of a “bad” edge is roughly the expected Ham-

7

Combinatorial Algorithms for Design of DNA Arrays

s

t (a)

(b)

Fig. 1. Threadings (shown by solid edges) of the same TSP tour on 4 ¥ 4 array: a standard row-

by-row threading and b 1-threading

ming distance between random probes since no optimization on these edges was done. However, it is not true for dashed edges in Fig. 1b.Although no explicit optimization on dashed edges was done, we implicitly minimized their cost while searching for a TSP tour since dashed edges join “close” vertices in the TSP tour (vertices i and i +3). An observation that the distance between close vertices in the TSP tour is generally lower than the average distance implies that the dashed edges correspond to relatively small distances. Therefore, some implicit optimization on these edges was done. Figure 1a has 3 dashed edges while Fig. 1b has 6 dashed edges thus indicating that threading in Fig. 1b is better. This observation leads to the following problem.

Fig. 2. k-Threading

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S. Hannenhalli et al.

Threading is a (self-avoiding) path p = p1 … pn through the elements of a grid with n cells. Notice that if pi and pj correspond to the neighboring cells then j–i is odd. Given a threading p and a threshold t, the vertices pi and pj in the threading are close (or t-close) if 1<|j–i|£t and the corresponding cells are neighbors in the grid. Optimal threading problem (OTP) is to find a threading maximizing the number of close vertices. Below we present the solution of the optimal threading problem for small values of t (t=3, 5, 7) that are adequate for arrays commonly used functional genomics. Define a k-threading as shown in Fig. 2. 0-threading is simply a row-by-row threading. If t = 1 then any threading (in particular, 0-threading) is optimal. It fact, we believe that the following conjecture is true: Conjecture k-threading solves the optimal threading problem for the threshold t= 2k+1. We proved this conjecture for t=3, 5, 7 and came up with rather tight bounds for k>7. Surprisingly, the proofs for all these three cases use different ideas. Theorem 1 1-Threading provides an optimal threading for OTP with the threshold 3. Proof We bound the number of close vertices in a threading p = p1 … pn . Assign to a threading p a sequence a=a1 …an such that ai =1 if pi is close to pi + 3 , and ai =0 otherwise. Simple geometric analysis demonstrates that if pi and pi+3 are close then pi+1 and pi+4 can not be close and pi–1 and pi+2 can not be close. Therefore, every 1 in the sequence a is preceeded and followed by a 0. Therefore, the number of 1’s in a (and the number of close vertices in the threading p) is at most n/2. On the other hand, the number of close vertices in 1-threading approaches n/2 thus proving that 1-threading is optimal. For a given threading p = p1 … pn and a threshold t define a graph on vertices {p1 , …, pn} with pi and pj adjacent iff they are either close or consecutive vertices in the threading (Fig. 3). For vertices pi and pi+2 k+1 define (pi+k , pi+k+1) as the middle edge between pi and pi+2k+1 . Every pair of close vertices in the threading graph imposes a unit load on the middle edge between them. For example, close vertices p1 and p6 impose a unit load on the edge (p3 , p4) in the threading graph (Fig. 3). Close vertices p2 and p5 impose another unit load on the edge (p3 , p4) thus imposing overall load 2 on this edge. The overall load of all close vertices of the threading in Fig. 3 is 12. Below we prove that for the threshold 5, the overall load of any threading is bounded by 2/3 n thus demonstrating that 2threading is asymptotically optimal for t=5. Theorem 2 2-Threading provides an optimal threading for OTP with the threshold 5.

Fig. 3. Threading graph of a 2-threading with the threshold t=5. Bold edges have load 2

Combinatorial Algorithms for Design of DNA Arrays

9

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S. Hannenhalli et al.

Fig. 4. Fragments of the threading graphs for two possible configurations of close vertices at

distance 5. Bold edges have load 2 or 1

Proof We prove that the overall load of every threading with threshold 5 is at most 2/3 n. Since the number of 5-close vertices in 2-threading is 2/3 n asymptotically it proves that 2-threading is optimal for this threshold. It is sufficient to prove that the overall load of every three consecutive edge is at most 2. Figure 4 shows two different configurations for close edges at distance 5. Simple geometric analysis demonstrates that for the first configuration two edges to the left and two edges to the right from the loaded edge have zero load. For the second configuration the edge to the left and the edge to the right from the pair of loaded edges have zero load. Therefore, every three consecutive edges have load at most 2. Theorem 3 3-Threading provides an optimal threading for OTP with the threshold 7. Proof The proof for t=7 is technically more involved. In this case, we have to prove that the number of edges in the threading graph is at most 3/4 n but the argument “every 4 edges have a load at most 3” does not work anymore (Fig. 5). We briefly sketch a different argument which proves the optimality of 3-threading in this case.A sequence of consecutive edges in the threading graph is a run if each edge

Fig. 5. An example of a threading graph with load 4 on four consecutive edges joining vertices 4 and 7 (t=7). Thick bold edges have load 2 while thin bold edges have load 1

11

Combinatorial Algorithms for Design of DNA Arrays

in this sequence has a non-zero load and the edges to the right and to the left of this sequence have a zero load. Every run with m edges has l>0 zero-load edges to the left and r>0 zero-load edges to the right. For each run define the parameter l+r p = m + 7 . It is easy to see that if for every run the parameter 2 load of the run 3 ≤3 998 p 4 then the number of close edges in the threading graph is smaller then 3/4 n. For example, for a run of edges (4, 5) and (5, 6) with loads 2 and 1 correspondingly, l=3, m=2, r=1. Therefore load of the run 2+1 3 = 751 ≤ 3 . 998 p 3+1 4 2+ 7 2 A case-by-case analysis of different configurations with t=7 demonstrates that load of the run 3 ≤3 998 p 4

for every run (proof is omitted). Table 3. Performance of 1-threading

1. Size 2. Chip Dimensions 3. A lower bound on a TSP tour cost 4. Local-greedy TSP tour cost 5. 2-opt improved TSP tour cost 6. Global-greedy TSP tour cost 7. Maximum % improvement in TSP tour cost 8. Achieved % improvement in TSP tour cost 9. Edge-Cost of 0-threading 10. Edge-Cost of 1-threading 11. Edge-Cost of improved 1-threading 12. Percentage decrease in Edge-cost 13. Flash-Cost of 0-threading 14. Flash-Cost of 1-threading 15. Percentage change in Flash-cost

DataSet 1 DataSet 2

DataSet 3 DataSet 4 DataSet 5

4K 134*134 28246

9K 134*134 57836

14K 256*256 96513

16K 256*256 60815

66K 262*262 221995

30045 30012 29367 5.9

61229 61187 60124 5.5

100675 100657 99108 4.1

64392 64213 63679 5.3

236835 235985 234356 5.9

2.1

1.7

1.5

0.8

0.7

92483 86764 86185

195959 181639 180653

308021 293652 291079

300392 253420 251761

1226529 1005429 993502

6.8

7.8

5.5

16.1

20.0

29639 35356 +19.2

60481 72939 +20.6

99207 118237 +19.2

62054 57190 –7.8

228823 208059 –9.1

12

S. Hannenhalli et al.

For the experimental data considered, t=3 seems the most appropriate, leading to 1-threading. Notice that this threading does not optimize the costs of vertical edges between the rows 2 i and 2i +1. To further improve this threading we apply the Lin-Kernighan [8] optimization on the pairs of rows of the grid (every vertex in the corresponding TSP is a pair of consecutive rows 2 i+1 and 2i). The improvement on this latest step is rather small (Table 1) thus indicating that 1-threading already provides a good solution.

5 Rectangle Cover Problem After the arrangement of oligonucleotides on the array is fixed, the resulting masks need to be generated by patterning equipment as unions of small rectangles (rectangle cover problem). In this section we describe the Rectangle_Cover algorithm that takes into account the specifics of oligonucleotide masks and leads to a 10%–30% improvement in the cover size as compared to a greedy strategy. We define a n¥n grid as a collection of n2 cells and 2n(n+1) edges. A mask is defined as a subset of the cells of a grid. Cells belonging to the mask are called black cells while the remaining cells are called white cells (Fig. 8). A rectangle is a set of black cells forming a rectangular shape. Given a mask, the rectangle cover problem is to find a minimum number of rectangles covering all black cells of the mask. An edge in a grid is a border edge for a mask if it separates a black cell from a white cell. A rectangle in a mask is maximal if each of its four sides contains a border edge. A black cell is called a leaf if it belongs to only one maximal rectangle in a mask. A maximal rectangle is prime if it contains a leaf. Two horizontal (vertical) edges are called co-horizontal (co-vertical) if they occupy the same column (row) of the grid. Each black cell x defines a maximal horizontal continuous strip of black cells containing x. This strip is bounded by covertical border edges left (x) on the left and right (x) on the right. The co-horizontal border edges top(x) and bottom(x) are defined similarly. For a cell x in a mask M a rectangle defined by border edges left(x), right(x), top (x) and bottom(x) is called the extended rectangle for x and is denoted R(x, M). Note that an extended rectangle might contain both black and white cells. The following lemma characterizes leaves and prime rectangles. Lemma 1 A cell x Œ M is a leaf iff R (x, M) contains only black cells. A rectangle in a mask M is prime iff it has a pair of co-horizontal and a pair of co-vertical border edges in M. Clearly, for every mask there exists an optimal cover which contains only maximal rectangles. It implies that there exists an optimal cover containing all prime rectangles. This observation motivates the first stage of the algorithm Rectangle_Cover (see [6]) for covering mask M (lines 1–2 in Fig. 6). The second stage (lines 3–11) is motivated by the following lemma.

Combinatorial Algorithms for Design of DNA Arrays

13

Fig. 6. Algorithm Rectangle_Cover

Lemma 2 Let be an optimal cover of mask M and let ´Ã . Define M´=M\»R Œ´ R. Let – R(x, M) be the extended rectangle of cell x Œ M´. If there exists a rectangle R – such that M´« R(x, M)Ã R ÃM, then there exists an optimal cover of M contain– ing all rectangles from ´»{R }. Lemma 2 ensures that a set of rectangles constructed by the algorithm Rectangle_Cover is contained in an optimal cover. For all oligonucleotide masks constructed at Affymetrix, is an optimal cover. To complete the cover construction for the cases when ´ is non-empty, we use a standard greedy Set_Cover [4] algorithm (although it was never invoked for real mask design). It is typical of oligonucleotide masks that prime rectangles either fully cover the mask (thus forming the optimal cover) or leave a small number of cells uncovered. In the latter case the uncovered cells usually form a very simple mask (frequently a collection of non-overlapping rectangles) and an optimal cover for this mask can be found using lemma 2 (second stage of the algorithm). Since the optimal cover for typical oligonucleotide masks is frequently found after the first stage of Rectangle_Cover we describe an efficient algorithm for generating maximal and prime rectangles in detail. Given the sets of intervals and define « ={ « : Œ and Œ }. Define

=(( « ) » ).

The dynamic programming algorithm Rectangle_List (Fig. 7) generates the set of all maximal rectangles for a n¥n mask M in O(n2) time. At the k-th iteration the Rectangle_List forms the set of all maximal rectangles positioned above the k-th row of M. M(k) in Rectangle_List is defined as the set of intervals

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Fig. 7. Algorithm Rectangle_List

corresponding to the k-th row of M, every such interval being a maximal continuous strip of black cells in the k-th row of M. A base of a rectangle is its lower side. It is easy to see that the bases of maximal rectangles cannot partially overlap thus implying that the number of maximal rectangles is O(n2). Therefore, Rectangle_List can be implemented in O(n2) time. Lemma 1 implies that an algorithm to generate the set of all prime rectangles can be implemented in O(n2) time thus leading to an O(n2) implementation of the first stage of Rectangle_Cover. Each iteration of the second stage of Rectangle_Cover can be implemented in O(n3). Since the number of such iterations is O(n2), the worst-case running time of the algorithm is dominated by the second stage and is O(n5). However, for oligonucleotide masks the number of the iterations of the second stage is very small (if any).

6 Mask Decomposition Problem Earlier attempts to minimize the flashcount generated partitions of the mask into rectangles (a set of non-overlapping rectangles covering the mask). Rectangle_Greedy which was used for mask design at earlier stages adds the largest possible rectangle to the already found partition while scanning the mask in left-toright top-to-bottom fashion. Due to the regular structure of standard mask designs, this algorithm proved to be very successful. An attempt to improve this algorithm resulted in the Rectangle_Partition algorithm that computes the partition complexity of a mask (i.e., the minimum number of non-overlapping rectangles covering a mask). Rectangle_Partition is based on a reduction of the rectangle partition problem to the maximum matching problem which was first devised for data compression in databases of twodimensional pictures [10]. Rectangle_Partition is based on a duality theorem which is given below. First we need a few definitions. Every mask consists of a connected areas and w internal windows. For example a mask in figure 1 consists of a=3 connected components and w=5 internal windows. Two points in a mask are outerconnected

Combinatorial Algorithms for Design of DNA Arrays

15

Fig. 8. Decomposition of a simple mask into rectangles

if there is a path between them that lies entirely outside the mask. For example, in Fig. 8 points 1 and 2 are outerconnected while points 1 and 12 are not. Every mask has two kinds of corners: sharp 90° degree corners and obtuse 270° degree corners. The mask shown in Fig. 8 has 22 sharp and 30 obtuse corners (obtuse corners are numbered in Fig. 8). A chord is a horizontal or vertical line joining two vertices on the (external or internal) border of the mask and having no other vertex on the border. Two obtuse corners are colinear if they have the same x or y coordinates and lie on the same chord. A mask is simple if no two obtuse corners are colinear. Figure 8 presents an example of a simple mask. The following theorem demonstrates that every obtuse corner and every connected component in the mask presents a potential obstacle while every window presents a potential bonus for decomposition of a simple mask.

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Theorem 4 The partition complexity of a simple mask with k obtuse corners is a– w+k. Proof Mask decomposition can be interpreted as drawing some chords in the mask (see Fig. 8). After a chord is drawn we assume that it creates a “strait” between two parts of the mask it separates.An important observation is that every chord joining vertices x and y increases the parameter a–w by exactly 1. If x and y are outerconnected than Da=1, Dw=0, if x and y are not outerconnected Da=0, Dw=–1 resulting in D(a– w)=1. After all m chords were drawn there is no windows left and a–w+m=c. Every chord in a simple mask can destroy at most one obtuse corner (since no two obtuse corners lay on the same chord) and in the end all obtuse corners should be destroyed (since there is no obtuse corner in a decomposition of the mask into rectangles). It implies that k ≤ m and a–w+k ≤ c. One can see that making k horizontal chords (one for each obtuse corner) destroys all obtuse corners and decomposes the simple mask into rectangles (Fig. 8). Therefore for a simple mask c=k+a–w. Theorem 4 works only for simple masks and fails as soon as a mask has colinear obtuse corners. For example, the mask in Fig. 9 has colinear obtuse corners (2, 13), (3, 9) and (4, 8) having the same x-coordinates and (1, 5), (15, 11), (14, 12) having the same y-coordinates. For the mask in Fig. 9 the formula k+a–w=19–1+1=c does not work since a decomposition of this mask into 15 rectangles is possible. However, the complexity of such masks can be efficiently computed by solving the maximum independent set problem in a bipartite graph. Recall that in a simple mask every chord can destroy at most one obtuse corner thus providing a bound a–w+k ≤ c. Since in an arbitrary mask a chord can destroy up to two obtuse corners we have a–w+k/2 ≤ c. This bound is over-optimistic, since in Fig. 9 a–w+k/2=1–1–19/2=10
Combinatorial Algorithms for Design of DNA Arrays

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Fig. 9. Chord graph and decomposition of mask into rectangles

Theorem 5 is the basis of the Rectangle_Partition algorithm that reduces mask decomposition to two classical graph-theoretical problems: computing the number of connected components in a graph (to compute a and w) and computing the size of maximal independent set in the bipartite graph. Rectangle_Cover was implemented and compared with Rectangle_Greedy and Rectangle_Partition. Recall that Rectangle_Greedy and Rectangle_Partition generate a partition of a mask into rectangles (a set of non-overlapping rectangles covering the mask). The tables 6 and 3 compares the algorithms for both real masks and randomly generated masks. The Rectangle_Cover algorithm produces

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up to a 30% improvement as compared to Rectangle_Greedy that was originally used for array design at Affymetrix.

7 Conclusions DNA arrays are devices of complexity approaching that of microprocessors. The manufacturing of such devices is a rich source of optimization problems. These problems lead directly to practical improvements in the process, without changing the machines used to manufacture the arrays. The algorithms for the Border Minimization Problem allow probes of higher quality to be manufactured for a given feature size, by minimizing the amount of light leakage. This directly improves the quality of the arrays. In particular, threading is an efficient means of exploiting existing algorithms for the Travelling Salesman Problem to produce an efficient solution to the Optimal Placement Problem. The recent work exploited the two-dimensional nature of the array directly. The algorithms for the Mask Decomposition Problem decrease the cost of mask fabrication, and thus the cost of arrays. In particular, they allow masks of higher density to be made with pattern generator technology. Even the simple greedy algorithm allowed pattern generator technology to be used for a relatively long span of time. However, given the rapid increase in density of arrays, current generations of masks are generated using beam technologies, which are not limited by mask complexity. Acknowledgement. We are grateful to David Johnson for advice on approximation algorithms

and software for large TSP instances.

8 References 1. Angluin D, Valiant LG (1979) Fast probabilistic algorithms for hamiltonian circuits and matchings. Journal of Computer Science and Systems 18:155 2. Chee M, Yang R, Hubbell E , Berno A, Huang XC, Stern D, Winkler J, Lockhart DJ, Morris MS, Fodor SA (1996) Accessing genetic information with high-density dna arrays. Science 274:610 3. Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, Lockhart DJ, Davis RW (1998) A genome-wide transcriptional analysis of the mitotic cell cycle. Molecular Cell 2:65 4. Cormen TH, Leiserson CE, Rivest RL (1990) Introduction to Algorithms. MIT Press 5. Fodor S, Read JL, Pirrung MC, Stryer L, Tsai Lu A, Solas D (1991) Light-directed, spatially addressable parallel chemical synthesis. Science 251:767 6. Franzblau DS, Kleitman DJ (1984) An algorithm for covering polygons with rectangles. Information and Control 63:164 7. Garey MS, Johnson DS (1979) Computers and Intractibility: A Guide to the Theory of NPCompleteness. W. H. Freeman, San Francisco, CA 8. Lin S, Kernighan BW (1973) An effective heuristic for the traveling-salesman problem. Operation Research 21:498

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9. Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, Mittmann M, Wang C, Kobayashi M, Horton H, Brown EL (1996) Expression monitoring by hybridization to highdensity oligonucleotide arrays. Nature Biotechnology 14:1675 10. Ohtsuki, T (1982) Minimum dissection of rectilinear regions. Proc. 1982 IEEE Symp. on Circuits and Systems 1210 11. Pevzner PA (2000) Computational Molecular Biology: An Algorithmic Approach. The MIT Press instead 12. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F (1999) Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nature Biotechnology 17:974 13. Wang DG, Fan J, Siao C, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris M, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson T, Lipshutz R, Chee M, Lander E (1998) Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 280:1077 14. Wodicka L, Dong H, Mittmann M, Ho MH, Lockhart DJ (1997) Genome-wide expression monitoring in saccharomyces cerevisiae. Nature Biotechnology 15:1539

Received: August 2001

CHAPTER 2

High-Density GeneChip Oligonucleotide Probe Arrays Glenn H. McGall 1 · Fred C. Christians 2 1

Affymetrix, Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA. E-mail: [email protected] 2 Affymetrix, Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA. E-mail: [email protected]

High-density DNA probe arrays provide a highly parallel approach to nucleic acid sequence analysis that is transforming gene-based biomedical research. Photolithographic DNA synthesis has enabled the large-scale production of GeneChip probe arrays containing hundreds of thousands of oligonucleotide sequences on a glass “chip” about 1.5 cm2 in size. The manufacturing process integrates solid-phase photochemical oligonucleotide synthesis with lithographic techniques similar to those used in the microelectronics industry. Due to their very high information content, GeneChip probe arrays are finding widespread use in the hybridization-based detection and analysis of mutations and polymorphisms (“genotyping”), and in a wide range of gene expression studies. Keywords: GeneChip array, Oligonucleotide probe array, Photolithography, Gene expression monitoring, Genotyping

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1

Introduction

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Array Production Technology . . . . . . . . . . . . . . . . . . . . 24

2.1 2.2 2.3 2.4

Substrate Preparation and General Approach Photolithography . . . . . . . . . . . . . . . Light-Directed Synthesis Chemistry . . . . . Future Enhancements . . . . . . . . . . . .

3

Applications

3.1 3.2 3.3

Gene Expression Monitoring . . . . . . . . . . . . . . . . . . . . . 31 Genotypic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 38

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

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Abbreviations CE DMT DLP

2-cyanoethyl 4,4¢ dimethoxytriphenylmethyl digital light processor Advances in Biochemical Engineering/ Biotechnology, Vol. 77 Managing Editor: T. Scheper © Springer-Verlag Berlin Heidelberg 2002

22 DTT HPLC kb MeNPOC NNEOC NPPOC NTP PAG PEO PIV pTs PYMOC SNP TCEP

G.H. McGall · F.C. Christians

dithiothreitol high performance liquid chromatography kilobase a-methyl-6-nitropiperonyloxycarbonyl 1-(8-nitronaphth-1-yl)ethyloxycarbonyl 2-(2-nitrophenyl)propyloxycarbonyl nucleoside 5¢-triphosphate photo-acid generator poly(ethylene oxide) pivaloate para-toluenesulfonate 1-pyrenylmethyloxycarbonyl single-nucleotide polymorphism tris(2-carboxyethyl)phosphine

1 Introduction High-density polynucleotide probe arrays are among the most powerful and versatile tools for accessing the rapidly growing body of sequence information that is being generated by numerous public and private sequencing efforts. Consequently, this technology is having a major impact on biological and biomedical research [1, 2]. These arrays are essentially large sets of nucleic acid probe sequences immobilized in defined, addressable locations on the surface of a substrate capable of accessing large amounts of genetic information from biological samples in a single hybridization assay. In a typical application [2], DNA or RNA “target” sequences of interest are isolated from a biological sample using standard molecular biology protocols. The sequences are fragmented and labeled with fluorescent molecules for detection, and the mixture of labeled sequences is applied to the array, under controlled conditions, for hybridization with the surface probes. The array is then imaged with a fluorescence-based reader to locate and quantify the binding of target sequences from the sample to complementary sequences on the array, and software reconstructs the sequence data and presents it in a format determined by the application. Thus, in addition to the arrays themselves, the Affymetrix GeneChip system provides a fluidics station for performing reproducible, automated hybridization and wash functions; a highresolution scanner for reading the fluorescent hybridization image on the arrays; and software for processing and querying the data (Fig. 1). GeneChip technology is distinguishable from other microarray methods in that oligonucleotide probe sequences are photolithographically synthesized, in a parallel fashion, directly on a glass substrate. In a minimum number of synthetic steps, arrays containing hundreds of thousands of different probe sequences, typically 20 or 25 bases in length, can be generated at densities in the order of 105 –106 sequences/cm2 (Fig. 2). This capability, deliverable on a commercial production scale, is well beyond that of any other technology currently

High-Density GeneChip Oligonucleotide Probe Arrays

23

Fig. 1. GeneChip system overview

Fig. 2. Wafer-scale GeneChip production specifications

available and allows unprecedented amounts of sequence information to be encoded in the arrays. Other array construction technologies, such as micropipetting or inkjet printing, rely on mechanical devices to deliver minute quantities of reagents to predefined regions of a substrate in a sequential fashion. In contrast, the photolithographic synthesis process is highly parallel in nature, making it intrinsically robust and scalable. This provides significant flexibility and cost advantages in terms of materials management, manufacturing throughput, and quality control. To researchers the benefits are a high degree of reliability, uniformity of array performance, and an affordable price.

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Fig. 3. Photolithographic synthesis of oligonucleotide arrays

2 Array Production Technology The advent of DNA array technology has relied on the development of methods for fabricating arrays with sufficiently high information content and density in a rapid, reproducible and economic fashion. Light-directed synthesis [3–7] has made it possible to manufacture arrays containing hundreds of thousands of oligonucleotide probe sequences on glass “chips” little more than 1 cm2 in size, and to do so on a commercial scale. In this process, 5′- or 3′-terminal protecting groups are selectively removed from growing oligonucleotide chains in predefined regions of a glass support, by controlled exposure to light through photolithographic masks (Fig. 3). 2.1 Substrate Preparation and General Approach

Prior to photolithographic synthesis, planar glass substrates are covalently modified with a silane reagent to provide a uniform layer of covalently bonded hydroxyalkyl groups on which oligonucleotide synthesis can be initiated (Fig. 4). In a second step, a photo-imagable layer is added by extending these synthesis sites with a poly(ethylene oxide) linker which has a terminal photolabile hydroxyl protecting group. When specific regions of the surface are exposed to light, synthesis sites within these regions are selectively deprotected, and thereby “activated” for the addition of nucleoside phosphoramidite building blocks. These nucleotide precursors, also protected at the 5′ or 3′ position with a photolabile protecting group, are applied to the substrate, where they react with the surface hydroxyl groups in the pre-irradiated regions. The monomer coupling step is carried out in the presence of a suitable activator, such as tetrazole or di-

High-Density GeneChip Oligonucleotide Probe Arrays

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Fig. 4. Chemical preparation of glass substrates for light-directed synthesis of oligonucleotide

arrays

cyanoimidazole. The coupling reaction is followed by conventional capping and oxidation steps, which also use standard reagents and protocols for oligonucleotide synthesis [5, 7].Alternating cycles of photolithographic deprotection and nucleotide addition are repeated to build the desired two-dimensional array of sequences, as described in Fig. 3. Semiautomated cleanroom manufacturing techniques, similar to those used in the microelectronics industry, have been adapted for the large-scale commercial production of GeneChip arrays in a multi-chip wafer format (Fig. 5). Each wafer contains 49–400 replicate arrays, depending on the size of the array,

Fig. 5. Automated array manufacturing

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and multiple-wafer lots can be processed together in a procedure that takes less than 24 h to complete. Multiple lots are processed simultaneously on independent production synthesizers operating continuously. After a final chemical deprotection, finished wafers are diced into individual arrays, which are finally mounted in injection-molded plastic cartridges for single-use application (see Fig. 1). 2.2 Photolithography

The photolithographic process provides a very efficient route to high-density arrays by allowing parallel synthesis of large sets of probe sequences. The number of required synthetic steps to fabricate an array is dependent only on the length of the probes, not the number of probes. A complete set, or any subset, of probe sequences of length “n” requires 4¥n synthetic steps, at most. Masks can be designed to make arrays of oligonucleotide probe sequences for a variety of applications. Most arrays are comprised of custom-designed sets of probes 20–25 bases in length, and optimized masking strategies allow such arrays to be completed in as few as 3n steps. The spatial resolution of the photolithographic process determines the maximum achievable density of the array and therefore the amount of sequence information that can be encoded on a chip of a given physical dimension. A contact lithography process (Fig. 3) is used to fabricate GeneChip arrays with individual probe features that are 18¥18 microns in size. Between 49 and 400 identical arrays are produced simultaneously on 5˝¥5˝ wafers. For the largestformat chip currently in production (1.6 cm2), this provides wafers of 49 individual arrays containing more than 400,000 different probe sequences each. For arrays containing fewer probe sequences, this feature size enables more replicate arrays to be fabricated on each wafer. The technology has proven capability for fabricating arrays with densities greater than 106 sequences/cm2, corresponding to features less than 10¥10 microns in size. This level of miniaturization is beyond the current reach of other technologies for array fabrication. 2.3 Light-Directed Synthesis Chemistry

The current manufacturing process employs nucleoside monomers protected with a photoremovable 5′-(α-methyl-6-nitropiperonyloxycarbonyl), or “MeNPOC” group [4, 5], depicted in Fig. 6, which offers a number of advantages for large-scale manufacturing. These phosphoramidite monomers are relatively inexpensive to prepare, and photolytic deprotection is induced by irradiation at near-UV wavelengths (f~0.05; lmax ~350 nm) so that photochemical modification of the oligonucleotides, which absorb energy at lower wavelengths, can be avoided. The photolysis reaction involves an intramolecular redox reaction and does not require any special solvents, catalysts or coreactants. Since the photolysis can be performed “dry”, high-contrast contact lithography can be used to achieve very high-resolution imaging. Complete photodeprotection requires less

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hn (365nm)

Fig. 6. Light-directed oligonucleotide synthesis cycle using MeNPOC photolabile phospho-

ramidite building blocks

than 1 min using filtered I-line (365 nm) emission from a commercial collimated mercury light source. Photochemical deprotection rates and yields for oligonucleotide synthesis can both be monitored directly on planar supports using procedures based on surface fluorescence [5]. We have also developed a sensitive assay in which test sequences are synthesized on a support designed to allow the cleavage and direct quantitative analysis of labeled oligonucleotide products using ion-exchange high performance liquid chromatography (HPLC) with fluorescence detection [8, 9]. This method involves photolithographic synthesis of test sequences after the addition of a base-stable disulfide linker and a fluorescein monomer to the support (Fig. 7). The disulfide linker remains intact throughout the synthesis and deprotection, but can be subsequently cleaved under reducing conditions to release the synthesis products, all of which are uniformly labeled with a 3′-fluorescein tag. The labeled oligonucleotide synthesis products are then analyzed using HPLC or capillary electrophoresis with fluorescence detection, enabling direct quantitative analysis of synthesis efficiency. The sensitivity of fluorescence is a key feature of this methodology, since the quantities of DNA synthesis products on flat substrates are very low (1–100 pmol/cm2 [9]), and difficult to analyze by other means. The average stepwise efficiency of the light-directed oligonucleotide synthesis process is limited by the yield of the photochemical deprotection step which, in the case of MeNPOC nucleotides, is 90%–94% [5]. The other chemical reactions involved in the base addition cycles (coupling, capping, oxidation) use reagents in a vast excess over surface synthesis sites, and, provided that sufficient reagent concentrations and time are allowed for completion, they are es-

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Fig. 7. Method for fluorescent labeling and cleavage of photolithographically synthesized

oligonucleotides allows quantitative analysis by HPLC

sentially quantitative. However, the sub-quantitative photolysis yields lead to incomplete or “truncated” probes, with the desired full-length sequences representing, in the case of 20mer probes, approximately 10% of the total synthesis products. For a number of reasons, the presence of truncated probe impurities has a relatively minor impact on the performance characteristics of arrays when they are used for hybridization-based sequence analysis. Firstly, the silanating agents used in this process provide an abundance of initial surface synthesis sites (>100 pmol/cm2), so that the final “concentration” of completed probes on the support remains high. Thus, each of the 20¥20 micron features on a typical array contains over 107 full-length oligonucleotide molecules (Fig. 2). It should be noted that there is an optimum surface probe density for maximum hybridization signal and discrimination. Thus, an increase in the synthetic yield through alternative chemistries or processes, while increasing the surface concentration of full-length probes, can actually reduce the quality of the hybridization data [10]. This is due to steric and electrostatic repulsive effects that result when oligonucleotide molecules are too densely spaced on the support. Secondly, ar-

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High-Density GeneChip Oligonucleotide Probe Arrays

ray hybridizations are typically carried out under stringent conditions so that hybridization to significantly shorter (
Improvements in Synthetic Yield. Several alternative photolabile protecting group chemistries have been described which may also be applicable to light-directed DNA array synthesis [9–15]. Some are capable of providing stepwise coupling yields in excess of 96%, and several examples are shown in Fig. 8. Achieving high synthetic yields with these alternative chemistries typically requires that a layer of solvent or catalyst be maintained over the substrate during the photodeprotection step. However, this has the drawback of adding significant complexity and cost to the manufacturing process. Furthermore, when solvent is required, the light image must be projected through the substrate from the reverse side, and image quality is somewhat degraded, thus limiting the achievable density of arrays that can be made this way. Nonetheless, certain array applications that have more stringent probe purity requirements could benefit substantially from improvements in photochemical synthesis yield. Such applications would include those in which, after hybridization, the probe-target duplexes are used as a platform for further reactions or analyses. For example, methods based on template-directed enzymatic probe extension, ligation or cleavage have been suggested as a means of improving allelic discrimination in hybridization-based mutation and polymorphism detection on arrays [16]. For this reason, we are currently developing a new generation of reagents for photolithographic synthesis that provide high synthetic yields without impacting the cost or lithographic performance of the current manufacturing process. Some of these biochemical assay formats will also require probe array synthesis to proceed in the 5′–3′ direction so that the probes will be attached to the support at the 5′-terminus. This can be achieved through the use of 3′-photo-activatable 5′-phosphoramidite building blocks [7].

PYMOC (refs. 11, 12)

NPPOC (refs. 13, 15)

NNEOC (ref. 9)

Fig. 8. Alternate photoremovable protecting groups for photolithographic oligonucleotide syn-

thesis

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G.H. McGall · F.C. Christians

Fig 9. DNA probe array synthesis using photoacid generation in a polymer film to remove acidlabile DMT protecting groups

Improvements in Photolithographic Resolution. In order to achieve higher spatial resolution, as well as synthetic yields and photolysis rates, we have developed novel photolithographic methods for fabricating DNA arrays which exploit polymeric photo-resist films as the photoimageable component [17–20]. One of the advantages of the photo-resist approach is that it can utilize conventional 4,4′dimethoxytrityl (DMT)-protected nucleotide monomers. These processes can also make use of chemical amplification of photo-generated catalysts to achieve higher contrast and sensitivity (shorter exposure times) than conventional photo-removable protecting groups. In this process, a polymeric thin film, containing a chemically amplified photo-acid generating (PAG) system, is applied to the glass substrate. Exposure of the film to light creates localized development of an acid catalyst in the film adjacent to the substrate surface, resulting in direct removal of DMT protecting groups from the oligonucleotide chains (Fig. 9). This process has provided stepwise synthetic yields >98%, photolysis speeds at least an order of magnitude faster than that achievable with photo-removable protecting groups, and photolithographic resolution capability well below 10 microns. This technology will enable the production of arrays with much higher information content than is currently attainable.

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Flexible Custom Array Fabrication. Another recent development has been the application of programmable digital micromirror devices, or “digital light processors” (DLPs), for photolithographic imaging, which offers a flexible approach to custom photolithographic array fabrication [21]. These devices were originally developed for digital image projection in consumer electronics products. They are essentially high-density arrays of switchable mirrors that reflect light from a source into an optical system that focuses and projects the reflected image. By using DLPs for photolithographic array synthesis, custom designs can be fabricated in a relatively short time, without the need for custom chrome-glass mask sets. This approach may offer an advantage to researchers who wish to vary designs frequently, and use only a small number of arrays of a given design. It should be noted that the standard lithographic approach using chrome-on-glass masks, which is ideal for mass production of standardized arrays, is also being adapted to the cost-effective production of smaller quantities of variable-content arrays. This is achieved through the use of high-throughput mask design and fabrication capabilities, combined with new strategies that dramatically reduce the number of masks required to synthesize custom arrays.

3 Applications GeneChip oligonucleotide probe arrays are used to access genetic information contained in both the RNA (gene expression monitoring) and DNA (genotyping) content of biological samples. Many different GeneChip products are now available for gene expression monitoring and genotyping complex samples from a variety of organisms. The ability to simultaneously evaluate tens of thousands of different mRNA transcripts or DNA loci is transforming the nature of basic and applied research, and the range of application of DNA probe arrays is expanding at an accelerating pace. Current information on Affymetrix products and specifications is available at the website (www.affymetrix.com/products).A number of representative applications of these arrays are discussed below. 3.1 Gene Expression Monitoring

Currently, the most popular application for oligonucleotide microarrays is in monitoring cellular gene expression. Standard GeneChip arrays are encoded with public sequence information, but custom arrays are also designed from proprietary sequences. Figure 10 depicts how a gene expression array interrogates each transcript at multiple positions. This feature provides more accurate and reliable quantitative information relative to arrays that use a single probe, such as a cDNA, PCR amplicon, or synthetic oligonucleotide for each transcript. Two probes are used at each targeted position of the transcript, one complementary (perfect match probe), and one with a single base mismatch at the central position (mismatch probe). The mismatch probe is used to estimate and correct for both background and signal due to non-specific hybridization. The number of transcripts evaluated per probe array depends on chip size, the individual probe

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A

B

Fig. 10. Gene expression monitoring with oligonucleotide arrays. A An image of a hybridized

1.28 ¥ 1.28 cm HuGeneFL array, with 20 probe pairs for each of approximately 5000 full-length human genes. B Probe design. To control for background and cross-hybridization, each perfect match probe is partnered with a probe of the same sequence except containing a central mismatch. Probes are usually 25mers, and are generally chosen to interrogate the 3′ regions of eukaryotic transcripts to mitigate the consequences of partially degraded mRNA. (Reprinted with permission from [6])

feature size, and the number of probes dedicated to each transcript. A standard 1.28¥1.28 cm probe array, with individual 18¥18 micron features, and 16 probe pairs per probe set, can interrogate over 20,000 transcripts. This number is steadily increasing as manufacturing improvements shrink the feature size, and

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as improved sequence information and probe selection rules allow reductions in the number of probes needed for each transcript. Arrays are now available to examine entire transcriptomes from a variety of organisms including several bacteria, yeast, drosophila, arabidopsis, mouse, rat, and human. Instead of monitoring the expression of a small subset of selected genes, researchers can now monitor the expression of all or nearly all of the genes for these organisms simultaneously, including a large number of genes of unknown function. Numerous facets of biology and medicine are being explored using this powerful new capability. Gene function has been explored by studying changes in expression levels throughout the cell cycle [22, 23]. Genetic pathways can be examined in great detail by monitoring the downstream transcriptional effects of inducing specific genes in cell culture, and the effects of drug treatment on gene expression levels can be surveyed [24]. Expression arrays have also been used to screen thousands of genes to identify markers for human diseases such as cancer [25], muscular dystrophy [26], diabetes [27], or for aging [28, 29]. One important area of research that is benefiting greatly from GeneChip technology is cancer profiling, wherein gene expression monitoring is used to classify tumors in terms of their pathologies and responses to therapy. Understanding the variation among cancers is the key to improving their treatment. For example, a prostate tumor may be essentially harmless for 20 years in one patient, while an apparently similar tumor in another patient can be fatal within several months. One patient’s lymphoma may respond well to chemotherapy while another will not. This variation of pathologies has motivated oncologists to assemble an impressive body of information to help classify tumors based on numerous histological, molecular, and clinical parameters. This has required a massive effort by thousands of highly skilled and dedicated scientists over the past few decades. It is clear, however, that there is a need for improvement in tumor classification in terms of both understanding variations among tumors, and assigning tumors to appropriate classes. Two recent studies demonstrate the utility of GeneChip technology to cancer classification. In the first report [30], expression levels were measured in colon adenocarcinomas and normal colon tissues, and patterns differentiating the two sample types were revealed by two-way clustering analysis. It was found that ribosomal proteins were expressed at consistently higher levels in tumors than in normal tissue, thereby providing a small set of characteristic markers with large expression differences. Muscle-specific genes provided another distinguishing set of markers. These genes were expressed at a higher level in normal tissue, probably because of the tissue composition of the samples, largely epithelial tissue for the tumors and mixed tissue for the normal. In addition to identifying these two small sets of genes with relatively large expression differences, the authors report that examination of large sets of genes with even small differences in expression could reliably classify a sample as tumor or normal. Although the confidence level of an individual gene might be low, extensive expression analysis using thousands of genes reveals subtle, systematic differences with high confidence. Such an expression profile database constructed from samples of known types would be useful for class prediction, that is, classifying additional unknown samples.

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In a second report [31], class predictions were tested directly using a database built from two different types of leukemia. The current clinical tests used to distinguish ALL (acute lymphoblastic leukemia) from AML (acute myeloid leukemia), while useful, are imperfect and painstaking. Proper identification of these cancer types is critical because the treatments they require are quite different. In the first set of experiments, the authors examined 27 ALL and 11 AML samples to create a gene expression profile database. Statistical analysis identified about 1100 genes (of the 6817 examined) that correlated well with the ALLAML class distinction. Many of the most highly correlated genes had been previously implicated in cancer. The 50 most informative genes were used to accurately cross-validate 36 of 38 of the original samples (the other 2 calls were uncertain), and were also good predictors for an independent set of leukemia samples (29 of 34 were called correctly; the other 5 were uncertain). The test on independent samples is especially convincing given the more varied nature of the samples: two different RNA isolation methods were used by different laboratories; some samples were isolated from peripheral blood, some from bone marrow; some AML patients were adults, and some were children. Thus, the panel of the 50 most informative genes served as a strong class prediction set, and the authors reported similar results using 10–200 genes. The authors point out, however, that for other sample groups the number of genes needed for accurate class prediction may vary, and that no single gene correlated completely with class type. Golub et al. [31] went on to address the issue of class discovery, that is, the identification of new cancer classes. The statistical tool called self-organizing maps was employed to determine whether the original data set could be subdivided beyond the ALL-AML categories. The AML samples again clustered together, but the ALL samples were now split into two groups that were subsequently shown by immunotyping to be of B-cell or T-cell origin. Although this ALL subdivision was previously known, the clustering analysis would have discovered it even if it had not been known. Expression profiling coupled with appropriate statistical analysis holds promise not only in cancer classification, but also by extension to many other areas of disease research and management. Transcription profile databases may be assembled from samples that differ by tissue source, disease state or progression, morbidity/mortality, response to drugs and other treatments, and countless other variables. New patterns may be revealed and disease classes refined or discovered. Patients may be more finely stratified in clinical trials so that the success of treatments can be better judged, and the expectation is that the diagnosis and treatment of disease will improve substantially as a result. 3.2 Genotypic Analysis

As the human genome project finishes the first complete blueprint of the human genome, there is tremendous interest in identifying the variations in DNA sequences between individuals and relating these variations to phenotypes. It is of particular interest to understand how subtle sequence differences are associated

High-Density GeneChip Oligonucleotide Probe Arrays

35

with disease. Oligonucleotide arrays are well suited to probe these variations, particularly single-nucleotide substitutions and, to a lesser degree, short deletions and insertions. Oligonucleotide arrays are currently used primarily for two types of genotyping analysis. Arrays for mutation or variant detection (Fig. 11) are used to screen sets of contiguous sequence for single-nucleotide differences. Given a reference sequence, the basic design of genotyping arrays is quite simple: four probes, varying only in the central position and each containing the reference sequence at all other positions, are made to interrogate each nucleotide of the reference sequence. The target sequence hybridizes most strongly to its perfect complement on the array, which in most cases will be the probe corresponding to the reference sequence, but, in the case of a nucleotide substitution, this will be one of the other three probes. Up to 50 kb of sequence can thus be determined with 200,000 probes, or 400,000 probes if both DNA strands are interrogated. Impending decreases in array feature size (see Sect. 2.4) will extend this capacity further. Mutation detection arrays have been used to analyze the entire 16.5 kb sequence of mitochondrial DNA samples [32], the 9.2 kb coding sequence of the ATM gene [33], BRCA1 coding mutations [34, 35], p53 mutations [36, 37], cytochrome P450 variants involved in drug metabolism [38], and HIV sequence variants [39, 40], among others. The last three arrays are commercial products currently available from Affymetrix. The other main type of genotyping performed with oligonucleotide arrays is SNP analysis, that is, the genotyping of biallelic single-nucleotide polymorA

B

Fig. 11. Resequencing array for sequence variation detection. A Each base of a given reference sequence is represented by four probes, usually 20mers, that are identical to each other with the exception of a single centrally located substitution (bold). Shown are probe sets targeted to two adjacent positions of the reference sequence. B The target sequence is determined by hybridization intensities, with the probe complementary to the target providing the strongest signal. (Reprinted with permission from Warrington JA et al (2000) In: Microarray biochip technology. Biotechniques Books, p 122)

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phisms. Because SNPs are the most common source of variation between individuals, they serve not only as landmarks to create dense genome maps, but also as markers for linkage and loss of heterozygosity studies. Large numbers of publicly available SNPs – over one million to date – have been found using dideoxy sequencing as well as mutation detection arrays [41–43]. In addition to mutation detection arrays, at least two other types of oligonucleotide arrays can be used for SNP analysis. The “HuSNP” assay allows nearly 1500 SNP-containing regions of the human genome to be amplified in just 24 multiplex PCRs and then hybridized to a single HuSNP array. The SNPs cover all 22 autosomes and the X chromosome. Many of the 1500 SNPs were discovered using mutation detection arrays [41]. The probe strategy for a SNP array is shown in Fig. 12. The probes for each SNP on the HuSNP array interrogate not only the two alleles of the SNP position, but also three or four positions flanking the SNP; the redundant data are of higher quality for the same reasons that the use of multiple probes improves gene expression monitoring array data. SNP arrays have also proven useful for loss of heterozygosity studies [44, 45].

Fig. 12. HuSNP array design. A A known biallelic polymorphism at position 0 is interrogated by a block of four or five probe sets (five in this example). Each probe set consists of four probes, a perfect match and a mismatch to allele A, and a perfect match and a mismatch to allele B. One probe set in a block is centered directly over the polymorphism “0”), and others are centered upstream (–4, –1) and downstream (+1, +4). B The sequences of the probe set centered over the polymorphism is shown. C Sample images of blocks showing homozygous A, heterozygous A/B, or homozygous B at the same SNP site. (Reprinted with permission from Warrington JA et al (2000) In: Microarray biochip technology. Biotechniques Books, p 122)

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Fig. 13. Schematic of the single-base extension assay applied to Tag probe arrays. Regions con-

taining known SNP sites (A or G in this example) are first amplified by PCR. The PCR product serves as the template for an extension reaction from a chimeric primer consisting of a 5′ tag sequence and a 3′ sequence that abuts the polymorphic site. The two dideoxy-NTPs that could be incorporated are labeled with different flurophors; in this example, ddUTP is incorporated in the case of the A allele, and ddCTP for the G allele. Multiple SBE reactions can be done in a single tube. The tag sequence, unique for each SNP, directs the extension products to a particular address on the Tag probe array. The proportion of a fluorophor at an address reflects the abundance of the corresponding allele in the original DNA. (Reprinted with permission from [45])

Although it is anticipated that the HuSNP assay will be appropriate for many applications, a more generic alternative is available in the form of the GenFlex array. For this array, 2000 20mer “tag” probe sequences were selected on the basis of uniform hybridization properties and sequence specificity. The array includes three control probes for each tag (a complementary probe and single-base mismatch probes for both the tag and its complement). One way to use the GenFlex array for SNP analysis is illustrated below (Fig. 13). In this example, a single-base extension reaction is used, in which a primer abutting the SNP is extended by one base in the presence of the two possible dideoxy-NTPs, each of which is labeled with a different fluorophor. Since each target-specific primer carries a different tag, the identity of each SNP is determined by hybridization of the single-base extension product to the corresponding tag probe in the GenFlex array [46]. The flexibility of the GenFlex approach lies in the freedom to partner any primer with any tag, a feature that enables other applications as well (Sect. 3.3). Unlike yeast or bacteria, the size and complexity of the human genome currently necessitates locus-specific amplification for these genotyping applications. Without amplification, the concentration of each target is simply too low. We are developing more general arrays and procedures to reduce sequence complexity

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while maintaining sufficient information content. This will eventually reduce or perhaps even eliminate the need to design, make, and handle large numbers of locus-specific primers and PCR products. 3.3 Other Applications

While oligonucleotide arrays have been used primarily to determine the composition of RNA or DNA, many other applications are possible as well. Any methodology that involves capturing large numbers of molecules that will hybridize to oligonucleotides can conceivably benefit from the highly parallel nature of these microarrays. Furthermore, the hybridized molecules, which are essentially libraries, can serve as a platform for subsequent analyses based on biochemical reactions. We describe below several recent “non-traditional” uses of GeneChip arrays, and suggest a number of other potential applications as well. Tag arrays, such as the GenFlex array mentioned in the preceding section, have been used as “molecular bar-code” detectors [47–49]. In these experiments, mixtures of multiple yeast strains – each carrying a unique tag in its genome and having a different gene deleted – were subjected to a test such as drug treatment or growth in minimal medium, and then tag probe arrays were used to determine the proportion of each strain in the surviving population. As in gene expression and genotyping applications, the molecular bar-coding strategy takes advantage of the ability of probe arrays to selectively bind many different sequences in a complex mixture simultaneously. Parallel processing is not only faster and easier – it also minimizes the effect of variations in sample handling, thereby increasing the accuracy and precision of the measurements. It is also envisioned that tag probe arrays will be useful for proteomics and other protein screening applications. For example, by attaching a different oligonucleotide sequence tag to each member of a group of proteins to be analyzed, hybridization would allow them to be arrayed in discrete locations on a chip for parallel screening. Proteins of interest would be identified by their position on the array. In one possible approach (Fig. 14), the tag is attached to the protein genetically by linking the tag to the mRNA and then translating the protein in such a manner that the protein remains associated with the mRNA, as is done in ribosome display to create and capture high affinity antibodies [50]. The protein-mRNA-tag complex is hybridized to the tag probe array, and screened for protein activity on the array. It is also conceivable that the proteins could be translated on the array, after hybridization. Genes of interest are recovered, either directly from the array or from another aliquot of the mRNA library, by PCR using the tag sequence for one primer and a common 3′ end sequence as the other primer. One use for such a system would be in directed evolution projects in which large gene libraries are made by cloning into cells, usually bacteria or yeast, followed by propagating and screening each clone individually for production of a protein with new or improved properties. The tag system would not only eliminate the need to transform and handle individual clones, but would also allow highly parallel screening because thousands of variants could be assayed simul-

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Fig. 14. Using Tag probe arrays to screen protein activity. To a protein-encoding mRNA a 5′ tag sequence and a 3′ ribosome-blocking sequence are attached (A). In a pool of such molecules, such as a randomly mutated gene library, each mRNA is paired with a unique tag and all have the same 3′ sequence. Following in-vitro translation either on a microarray or in a test tube, the nascent protein remains attached to the mRNA (B). During hybridization the tag directs each mRNA-protein to a particular address on the Tag probe array (C), where all the proteins are screened simultaneously for activity (D). Appropriate detection methods identify proteins of interest (E, black and/or shaded blocks). Finally, the corresponding genes can be captured by PCR of the mRNA pool using a universal reverse primer and each identified Tag sequence as a forward primer

taneously on the same array. Another use for the tag system would be to screen (poly)peptides made from existing mRNA molecules for properties such as drug binding. For example, all the mRNAs from a pathogenic bacterial strain could be converted to tagged proteins, which could then be screened for the ability to bind antibiotic candidates. The RNA molecules themselves could also be screened, as some drugs act directly on RNA. It is also conceivable that the oligonucleotide tag could be added directly to proteins, a method that might be useful in cases in which clones are already separated and one wishes to use the tag probe array only for parallel screening. Researchers have also found a variety of novel uses for GeneChip arrays that were originally designed for gene expression monitoring. For example, Cho and co-workers [51] carried out a yeast two-hybrid study of S. cerevisiae proteins, mixing DNA from positive clones and hybridizing them to a yeast expression array, enabling them to identify the clones much more efficiently than if they had sequenced the clones by traditional methods. Winzeler et al. [52] used yeast expression arrays to identify more than 3700 biallelic genomic variations between

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two yeast strains and then used the markers to simultaneously map five loci with high resolution (11–64 kb). Deletions in yeast [53] and in a clinical M. tuberculosis strain [54] were identified by similar techniques, a potentially important application given the propensity of some pathogens to avoid drug and vaccination treatments by deleting segments of their genomes. The use of multiple probes for each gene, a characteristic feature of GeneChip expression arrays, was critical to the high degree of resolution that was achieved in these experiments. Milner et al. [55] reported using oligonucleotide arrays to survey oligonucleotide binding to a specific mRNA. A prevalent approach in anti-gene therapeutics involves knocking out malfunctioning genes through RNase H-mediated degradation of the mRNA, induced by duplex formation with antisense oligonucleotides. Presumably, the ability to predict, or at least empirically select, oligonucleotides which hybridize best to a given mRNA would be useful in the development of anti-gene therapeutics. Gene expression arrays have also served as platforms for the analysis of splice variation in organisms with introns, and for mapping transcriptional boundaries [56].Also, samples can be preselected for certain properties before hybridization, and at least two examples of this have been reported. In one case, cellular RNA [57], and in the other cellular DNA [58], were mixed with specific proteins, and complexes were purified by immunoprecipitation. Hybridization of the nucleic acids from the purified complexes revealed specific associations with the proteins. These two elegant experiments were carried out using arrays of spotted PCR products, but, again, one would expect that data of even higher resolution would be achievable using the multi-sequence probe sets present on GeneChip expression monitoring arrays. One could conceivably treat hybridized expression arrays with RNase H, and assay for activity directly by following the loss of signal. This type of approach could be useful for revealing potential RNase H “hotspots” in mRNAs. A number of other powerful, but as yet under-utilized, applications also use arrayed probetarget duplexes as a platform for further reactions or analyses [59]. For example, Bulyk, Church and co-workers [60] created arrays of four base-pair restriction enzyme recognition sites and demonstrated activity with the appropriate enzymes, including dam methylase. Studies such as this provide further demonstration that arrays of double-stranded probes offer a promising platform for studying DNA-protein interactions. Methods based on template-directed enzymatic probe extension, ligation or cleavage are also being investigated as a means of improving allelic discrimination in hybridization-based mutation and polymorphism detection on arrays [16]. It is expected that hybridization-based biochemical assays on DNA microarrays will become increasingly commonplace in the coming years, especially in the area of high-throughput genotyping applications.

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4 References 1. Phimister B (ed) (1999) Nat Genet Suppl 21:1 2. Schena R, Davis RW (2000) In: Schena M (ed) Microarray biochip technology. BioTechniques Books, Natick, MA, p 1 3. Fodor SPA, Read JL, Pirrung MC, Stryer LT, Lu A, Solas D (1991) Science 251:767 4. Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, Fodor SPA (1994) Proc Natl Acad Sci USA 91:5022 5. McGall GH, Barone AD, Diggelmann M, Fodor SPA, Gentalen E, Ngo N (1997) J Am Chem Soc 119:5081 6. Lipshutz R, Fodor SPA, Gingeras TR, Lockhart DJ (1999) Nat Genet Suppl 21:20 7. McGall GH, Fidanza JA (2001) In: Rampal JB (ed) Methods in molecular biology. DNA arrays methods and protocols. Humana Press, Inc., Totowa, NJ, p 71 8. McGall GH, Barone AD, Diggelmann M (1999) Eur Pat Appl EP 967,217 9. Barone AD, Beecher JE, Bury P, Chen C, Doede T, Fidanza JA, McGall GH (2001) Nucleosides Nucleotides 20:525 10. Unpublished data 11. McGall GH (1997) In: Hori W (ed) Biochip arrays. IBC Library Series, Southboro, MA, p 2.1 12. McGall GH, Nam NQ, Rava R (2000) US Patent 6,147,205 13. Hasan A, Stengele K-P, Giegrich H, Cornwell P, Isham KR, Sachleben R, Pfleiderer W, Foote RS (1997) Tetrahedron 53:4247 14. Pirrung MC, Fallon L, McGall G (1998) J Org Chem 63:241 15. Beier M, Hoheisel JD (2000) Nucleic Acids Res 28:e11 16. Tonisson N, Kurg A, Lohmussaar E, Metspalu A (2000) In: Schena M (ed) Microarray biochip technology. BioTechniques Books, Natick, MA, p 247 17. McGall G, Labadie J, Brock P, Wallraff G, Nguyen T, Hinsberg W (1996) Proc Natl Acad Sci USA 93:13555 18. Wallraff G, Labadie J, Brock P, Nguyen T, Huynh T, Hinsberg W, McGall G (1997) Chemtech, February:22 19. Beecher JE, McGall GH, Goldberg MJ (1997) Preprints Am Chem Soc Div Polym Mater Sci Eng 76:597 20. Beecher JE, McGall GH, Goldberg MJ (1997) Preprints Am Chem Soc Div Polym Mater Sci Eng 77:394 21. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F (1999) Nat Biotechnol 17:974 22. Cho RJ, Campbell MJ, Winzeler EA, Steinmetz L, Conway A, Wodicka L, Wolfsberg TG, Gabrielian AE, Landsman D, Lockhart DJ, Davis RW (1998) Mol Cell 2:65 23. Cho RJ, Huang M, Dong H, Steinmetz L, Sapinoso L, Hampton G, Elledge SJ, Davis RW, Lockhart DJ, Campbell MJ (2001) Nat Genet 27:48 24. Debouck C, Goodfellow PN (1999) Nat Genet Suppl 21:48 25. Liotta L, Petricoin E (2000) Nat Rev Genet 1:48 26. Chen YW, Zhao P, Borup R, Hoffman EP (2000) J Cell Biol 151:1321 27. Wilson SB, Kent SC, Horton HF, Hill AA, Bollyky PL, Hafler DA, Strominger JL, Byrne MC (2000) Proc Natl Acad Sci USA 97:7411 28. Lee CK, Klopp RG, Weindruch R, Prolla TA (1999) Science 285:1390 29. Ly DH, Lockhart DJ, Lerner RA, Schultz PG (2000) Science 287:2486 30. Alon U, Barkai N, Notterman DA, Gish K,Ybarra S, Mack D, Levine AJ (1999) Proc Natl Acad Sci USA 96:6745 31. Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing JR, Caligiuri MA, Bloomfield CD, Lander ES (1999) Science 286:531 32. Chee M,Yang R, Hubbell E, Berno A, Huang XC, Stern D,Winkler J, Lockhart DJ, Morris MS, Fodor SP (1996) Science 274:610

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33. Hacia JG, Sun B, Hunt N, Edgemon K, Mosbrook D, Robbins C, Fodor SP, Tagle DA, Collins FS (1998) Genome Res 8:1245 34. Hacia JG, Brody LC, Chee MS, Fodor SP, Collins FS (1996) Nat Genet 14:441 35. Hacia JG (1999) Nat Genet 21:42 36. Ahrendt SA, Halachmi S, Chow JT, Wu L, Halachmi N, Yang SC, Wehage S, Jen J, Sidransky D (1999) Proc Natl Acad Sci USA 96:7382 37. Wikman FP, Lu ML, Thykjaer T, Olesen SH, Andersen LD, Cordon-Cardo C, Orntoft TF (2000) Clin Chem 46:1555 38. Liu WW,Webster T,Aggarwal A, Pho M, Cronin M, Ryder T (1997) Am J Hum Genet 61:1494 39. Kozal MJ, Shah N, Shen N, Yang R, Fucini R, Merigan TC, Richman DD, Morris D, Hubbell E, Chee M, Gingeras TR (1996) Nat Med 2:753 40. Gunthard HF, Wong JK, Ignacio CC, Havlir DV, Richman DD (1998) AIDS Res Hum Retroviruses 14:869 41. Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lander ES, et al (1998) Science 280:1077 42. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Shaw N, Lane CR, Lim EP, Kalyanaraman N, Nemesh J, Ziaugra L, Friedland L, Rolfe A,Warrington J, Lipshutz R, Daley GQ, Lander ES (1999) Nat Genet 22:231 43. Lindblad-Toh K, Winchester E, Daly MJ, Wang DG, Hirschhorn JN, Laviolette JP, Ardlie K, Reich DE, Robinson E, Sklar P, Shah N, Thomas D, Fan JB, Gingeras T, Warrington J, Patil N, Hudson TJ, Lander ES (2000) Nat Genet 24:381 44. Lindblad-Toh K, Tanenbaum DM, Daly MJ, Winchester E, Lui WO,Villapakkam A, Stanton SE, Larsson C, Hudson TJ, Johnson BE, Lander ES, Meyerson M (2000) Nat Biotechnol 18:1001 45. Mei R, Galipeau PC, Prass C, Berno A, Ghandour G, Patil N, Wolff RK, Chee MS, Reid BJ, Lockhart DJ (2000) Genome Res 10:1126 46. Fan JB, Chen X, Halushka MK, Berno A, Huang X, Ryder T, Lipshutz RJ, Lockhart DJ, Chakravarti A (2000) Genome Res 10:853 47. Shoemaker DD, Lashkari DA, Morris D, Mittmann M, Davis RW (1996) Nat Genet 14:450 48. Giaever G, Shoemaker DD, Jones TW, Liang H,Winzeler EA,Astromoff A, Davis RW (1999) Nat Genet 21:278 49. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Davis RW, et al (1999) Science 285:901 50. Hanes J, Jermutus L, Pluckthun A (2000) Methods Enzymol 328:404 51. Cho RJ, Fromont-Racine M,Wodicka L, Feierbach B, Stearns T, Legrain P, Lockhart DJ, Davis RW (1998) Proc Natl Acad Sci USA 95:3752 52. Winzeler EA, Richards DR, Conway AR, Goldstein AL, Kalman S, McCullough MJ, McCusker JH, Stevens DA, Wodicka L, Lockhart DJ, Davis RW (1998) Science 281:1194 53. Winzeler EA, Lee B, McCusker JH, Davis RW (1999) Parasitology 118:S73 54. Salamon H, Kato-Maeda M, Small PM, Drenkow J, Gingeras TR (2000) Genome Res 10:2044 55. Milner N, Mir KU, Southern EM (1997) Nat Biotechnol 15:537 56. Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S (2001) Genes Dev 15:1637 57. Takizawa PA, DeRisi JL, Wilhelm JE, Vale RD (2000) Science 290:341 58. Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA (2000) Science 290:2306 59. Gunderson KL, Huang XC, Morris MS, Lipshutz RJ, Lockhart DJ, Chee MS (1998) Genome Res 8:1142 60. Bulyk ML, Gentalen E, Lockhart DJ, Church GM (1999) Nat Biotechnol 17:573 Received: June 2001

CHAPTER 3

Oligonucleotide Scanning Arrays: Application to HighThroughput Screening for Effective Antisense Reagents and the Study of Nucleic Acid Interactions M. Sohail · E.M. Southern University of Oxford, Department of Biochemistry, South Parks Road, Oxford OX1 3QU, England, UK. E-mail: [email protected]

Oligonucleotide scanning arrays are useful tools in the study of nucleic acid interaction. Such arrays of oligonucleotides, corresponding to a full set of complements of a known sequence, can be readily made in a single series of coupling reactions, adding each nucleotide in the complement of the target sequence in turn. The synthesis is carried out on the surface of a solid substrate such as glass or polypropylene that has been modified to allow nucleotide coupling. A mask is used to apply the DNA synthesis reagents in a defined area and is moved by a fixed step size after each coupling reaction so that consecutive couplings overlap a portion of the previous one. The size of the mask and the displacement at each coupling determine the length of the oligonucleotides. A radiolabeled or fluorescently tagged target sequence is hybridised to a scanning array and its interaction with the complementary oligonucleotides is displayed as a hybridisation signal. It is thus possible to determine the exact sequence and lengths of large numbers of interacting sequences in a single hybridisation experiment. The array image is analysed using a computer program (xvseq) that calculates quantitative measurements of the binding strengths.We have found scanning arrays a useful tool not only to find effective antisense reagents, but also to study RNA folding and the mechanisms of heteroduplex formation. In this article, we discuss the format of these arrays, the technology used to fabricate and to read them, and their applications.

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1

Introduction

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The Format of Scanning Arrays . . . . . . . . . . . . . . . . . . . 44

3

Making and Reading Scanning Arrays

3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.2 3.3

Fabrication . . . . . . . . . . . . . . . . . . . . . . . . . Solid Supports for Making Arrays . . . . . . . . . . . . . Masks for Making Arrays: Materials and Machining . . . Making Scanning Arrays on an ABI DNA/RNA Synthesiser Deprotection of Arrays . . . . . . . . . . . . . . . . . . . Hybridisation of a Labeled Target to Scanning Arrays . . Reading a Scanning Array Image . . . . . . . . . . . . .

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Applications

4.1 4.2

High-Throughput Screening of Antisense Reagents . . . . . . . . 53 The Study of Nucleic Acid Folding and Heteroduplex Formation . . 54

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47 47 48 48 50 51 52

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4.2.1 4.2.2 4.2.3

RNA Folding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Heteroduplex Formation . . . . . . . . . . . . . . . . . . . . . . . 55 A Study of the Secondary Structure of Nucleic Acids with Modified Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

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Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . 56

6

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

1 Introduction Scanning arrays are made by combinatorial in situ synthesis of a large number of oligonucleotides on a solid support in a spatially addressable fashion. They comprise sets of oligonucleotides of various lengths in which a series of oligonucleotides, complementary to the target RNA or DNA, is made by sequential coupling of nucleotides to a solid substrate (such as glass or polypropylene) that has been modified to allow oligonucleotide synthesis. The set of oligonucleotides covers every nucleotide position in the region of interest of the complement of the target. The resultant array allows an exhaustive and parallel analysis of the interactions of a complete set of oligonucleotides complementary to the target under investigation in a single hybridisation experiment (see flow chart in Fig. 1).

2 The Format of Scanning Arrays The process of array fabrication allows the synthesis of oligonucleotides that differ from the adjacent members in the set by one or two nucleotides and they are in the order in which they occur in the complement of the target sequence. If the mask used to make arrays is kept in the same place during all the synthesis cycles, the result will be a complete complement of the target sequence. However, if the mask is moved along the surface after each nucleotide coupling, the result is a series of oligonucleotides, each one complementary to a region of the target sequence. The displacement of the mask is such that the mask overlaps a portion of its footprint at the previous position. The oligonucleotides range in size from monomers to a certain maximum, generally around 20–25 residues. The longest in the set of the oligonucleotides is determined by the size of the mask used to retain DNA synthesis reagents and the displacement of the mask after each nucleotide coupling (Fig. 2). We generally use either diamond-shaped or circular masks to make scanning arrays. For a diamond-shaped mask, the length of the longest oligonucleotide is the ratio of the diagonal of the mask to the displacement and for a circular mask it is the ratio of the diameter of the circle to the displacement at each nucleotide coupling. For example, with a diamond-shaped mask of 30 mm diagonal, a displacement of 2 mm at each addition will result in 15-mers along the centre line, while a displacement of 1.5 mm will result in 20-mers. The cells created by over-

Oligonucleotide Scanning Arrays

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Fig. 1. Flow chart summarising the process of scanning array farication, hybridisation and

imaging

in front of a DNA synthesiser. The lead screw is fitted with a pusher nut that drives the plate across the front of the mask by driving the turning wheel. The pressure clamp is fixed to the back of the frame with a polyethylene cushion mounted on its pressure pad. In the automated version of the array maker, both the turning wheel and the pressure clamp have been replaced with computer-controlled motors. B A diamond-shaped mask. The mask consists of a block (metal or PTFE) with a diamond-shaped sealing edge. Two holes, drilled just inside of the top and the bottom corners (marked inlet and outlet) of the diamond shape, can take shortened 19SWG syringe needles and are used to connect the mask to the reagents supply of the DNA synthesiser. C Format of scanning arrays. A template grid of overlapping diamonds is shown. The longest oligonucleotides in the set are 10-mers (closed diamonds) located along the centre line of the grid. The positions of three 10-mers (1–10, 11–20 and 17–26) are marked on the top of the grid and the lengths of the oligonucleotides from monomers to 10-mers with common 3′- and common 5′-ends at the bottom. The arrays are symmetrical above and below the centre line allowing duplicate hybridisation measurements. Each smaller diamond-shaped cell is occupied by a single oligonucleotide sequence

Fig. 2. A Apparatus to make scanning arrays by hand. The mask shown in B is fixed on the frame and the frame (made from angled aluminium) is fixed

46 M. Sohail · E.M. Southern

Oligonucleotide Scanning Arrays

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lapping footprints of the diamond-shaped mask are small diamonds but the cells created by a circular mask differ in shape: along the centre line they are lenticular but off this line they form a four-cornered “spearhead” that diminishes in size towards the edge. Each of these smaller cells contains a single oligonucleotide sequence that differs from oligonucleotides in the neighbouring cells by one or two nucleotides, depending on its position (see Fig. 2). The longest oligonucleotides are found along the centre line of the array and monomers are located along the edges. In between are all lengths ranging from monomers to the longest in the set. The number of oligonucleotides of a particular length can be calculated by a simple formula, N–s+1, where s is the length of the oligonucleotides and N is the total number of nucleotide couplings used to make the array. For example, if a 100 nt long sequence is covered in 100 coupling steps (with a diamond-shaped mask of 30 mm diagonal and 1.5 mm step size), there will be 100 monomers, 96 5-mers, 91 10-mers, and 81 20-mers. The last 19 positions in sequence will be represented by shorter oligonucleotides only, from 19-mers to monomers in this case. Therefore, to make 100 20-mers in the array, an additional 19 nt synthesis steps must to be added at the end (total coupling steps = N+s–1). The arrays as fabricated are symmetrical above and below the centre line, thus each oligonucleotide is represented twice on the array allowing for duplicate hybridisation measurement providing an internal hybridisation control.Additionally, each half can be hybridised separately. In this way, one can measure the hybridisation of two types of targets to the same set of oligonucleotides under identical conditions, or study the interaction of the same target with oligonucleotides under different hybridisation conditions, allowing a direct comparison between two experiments.

3 Making and Reading Scanning Arrays 3.1 Fabrication 3.1.1 Solid Supports for Making Arrays

A flat and impermeable surface is required for in situ synthesis of oligonucleotides and both glass and polypropylene meet these criteria. Glass has a number of favourable properties including its easy availability, smooth surface, physical and chemical stability, and compatibility with the use of radiolabeled and fluorescently labeled targets. We have found that 3 mm window glass is a good material for array fabrication. To make the glass surface ready for chemical reaction with nucleotide precursors (standard nucleotide CE phosphoramidites), it is chemically derivatised with a 20-atom aliphatic chain to produce free hydroxyl groups on its surface [1]. Glass is first treated with a solution of glycidoxypropyl trimethoxysilane that reacts with the silanol groups on its surface to produce an epoxide group. This is then reacted with a polyethylene glycol leaving a linker/spacer molecule terminating in a hydroxyl group that is a sub-

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strate for reaction with nucleotide phosphoramidite [1]. Alternatively, the glass can be derivatised by exposing it to a vapour phase of trimethoxysilane in a vacuum furnace at approximately 140–145 °C for several hours. This creates a monolayer of the epoxide group on the surface that can then be reacted with polyethylene glycol. Polypropylene also has favourable physical and chemical properties. Its surface is aminated by plasma discharge in the presence of anhydrous ammonia [2]. The free amine groups (–NH2) thus produced on its surface are also a substrate for oligonucleotide synthesis with standard phosphoramidite chemistry. 3.1.2 Masks for Making Arrays: Materials and Machining

Both circular and diamond-shaped masks can be made from stainless steel, aluminium or PTFE (Teflon): the process of making them may differ with the type of the material used and the shape of the mask. Circular masks are made using a centre lathe and can be made from both metals and PTFE. Diamond-shaped masks are rather difficult to make with PTFE by the machining process but can be made by pressure moulding in a hydraulic press (~150 tonne force at 380 °C) using a pre-machined die of appropriate size. To make a diamond-shaped mask from metal (Fig. 2), the workpiece is held at an angle of 45° to the axis of the bed of the milling machine such that the diagonal of the mask runs parallel to the axis of the bed. The cavity is made by machining to the required depth (0.5–0.75 mm) to create a reaction chamber. The sealing edge (0.3–0.5 mm wide) is formed by machining the outlands to a depth of approximately 0.5 mm and is finished by polishing flat with successively finer grades of wetted abrasive paper from ~P600–P1200 (3M Inc., USA). The final polish is achieved by using a polishing grade crocus paper (A. J. Naylor & Co, England). Holes for reagent inlet and outlet are drilled, respectively, at the bottom and the top of the reaction chamber (in the corners of the diamond). For masks made with PTFE, the holes should be 1.0 mm diameter: for aluminium or stainless steel, the holes should be 1.07–1.08 mm diameter. Inlet and outlet connections to the synthesiser are made using standard 19SWG syringe needles (1.1 mm diameter) with the chamfered ends ground and de-burred. With these dimensions, a leaktight seal is usually produced without the need for an additional sealer. 3.1.3 Making Scanning Arrays on an ABI DNA/RNA Synthesiser

The arrays are made on an adapted ABI DNA synthesiser using standard nucleotide CE phosphoramidites. The process of making arrays has been described in detail previously [3]. Briefly it is as follows: The mask is fixed to the frame (Fig. 2) and the inlets and outlets are connected to the reagent supply and waste lines of the DNA synthesiser using syringe needles. The mask is sealed against glass or polypropylene substrate by applying pressure (for example with a Gclamp), creating a cavity of the shape of the mask through which the reagents for DNA synthesis flow. DNA synthesis thus proceeds over a confined area on the

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Table 1. Modified program for an ABI394 DNA/RNA synthesiser to deliver reagents for one

coupling cycle Step No.

Function No.

Function

Step time a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

106 103 64 42 2 1 101 111 58 34 33 34 33 34 33 34 103 64 2 1 42 2 63 41 64 1 103 2 1 64 42 2 42 2 42 2 42 2 1 62 40 103 1 64 42 2 42 2 1 107

Begin Wait 18 to waste 18 to column Reverse flush Block flush Phos prep Block vent Tet to waste Tet to column B+tet to column Tet to column B+tet to column Tet to column B+tet to column Tet to column Wait 18 to waste Reverse flush Block flush 18 to column Reverse flush 15 to waste 15 to column 18 to waste Block flush Wait Reverse flush Block flush 18 to waste 18 to column Reverse flush 18 to column Reverse flush 18 to column Reverse flush 18 to column Reverse flush Block flush 14 to waste 14 to column Wait Block flush 18 to waste 18 to column Reverse flush 18 to column Reverse flush Block flush End

999.0 5.0 30.0 8.0 5.0 3.0 2.0 1.7 1.0 3.0 1.0 3.0 1.0 3.0 1.0 75.0 5.0 10.0 5.0 15.0 8.0 5.0 15.0 5.0 5.0 20.0 10.0 5.0 5.0 12.0 8.0 12.0 8.0 12.0 8.0 12.0 8.0 3.0 5.0 35.0 20.0 5.0 5.0 25.0 10.0 25.0 10.0 3.0

a

Step times are for a diamond-shaped mask having 0.73 mm depth ¥30 mm diagonal and have to be adjusted for each mask.

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surface of the solid substrate.After a nucleotide has been added, the mask is displaced by a pre-determined step size, by moving the solid substrate in front of the mask in an appropriate horizontal direction. The movement is such that the back of the mask overlaps a portion of the front of its previous footprint. The mask is sealed against the substrate again for addition of the next base in the sequence. The first longest oligonucleotide in the set is made when the mask intersects its first footprint for the last time. Routinely, we make scanning arrays covering around 150 nt of the complement of the target sequence. The process of array fabrication has now been automated; the movement of the solid substrate along the mask and the opening and closing of the mask against the substrate are operated by computer-controlled motors. This should, in principle, allow fabrication of longer arrays scanning the complement of an entire target sequence. The cycle used to make arrays is slightly modified from that used with columns. An example of such a cycle is given in Table 1. The flow times depend on the size and the depth of the mask. It is difficult to monitor the efficiency of coupling from trityl yield due to the large amounts of reagents used in the synthetic process. However, several criteria suggest a high stepwise yield. First, the ratio of coupling reagents to the substrate is more than 200-fold greater in this process than in conventional solid-phase synthesis on a controlled pore glass (CPG) column. Secondly, the synthesis of any oligonucleotide on the array is dependent on successful coupling of each nucleotide. For example, each nucleotide in a 20-mer on the array occurs in 19 other 20-mers. Thus, successful synthesis of any one oligonucleotide (indicated by a positive hybridisation signal) will indicate successful synthesis in these others. Thirdly, synthesis of oligonucleotides using a cleavable linker and analysis by gel electrophoresis has suggested efficient yields (S. C. Case-Green and E. M. Southern, unpublished data). It is also possible to make arrays of oligonucleotides with several other compatible chemistries. For example, with the use of standard nucleotide CE phosphoramidites in synthesis, the oligonucleotides are attached to the solid support at their 3′-end; reverse phosphoramidites (Glen Research) can be used to make oligonucleotides that are attached at their 5′-end. Such orientation may be important in applications requiring enzymatic manipulations (e. g., nucleotide addition using a DNA polymerase). Iodine is used as an oxidising agent to produce phosphodiester bonds; this can be replaced with a sulfurising agent (Beaucage reagent) to produce phosphorothioate bonds in the arrayed oligonucleotides. Modified nucleotides (e.g., 2′O-methyl-modified) can be used to obtain other variations on the standard format. 3.1.4 Deprotection of Arrays

When using standard phosphoramidite chemistry to make oligonucleotides, the exocyclic amines of the bases are protected chemically to prevent side reactions from occurring during synthesis. These protecting groups must be removed from the coupled bases before hybridisation. We achieve this by either of the following two methods: (1) by leaving the arrays in an aqueous solution of ammonia

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at 55 °C for 14–16 h in a closed chamber, or (2) by incubating the array in an equal volume mixture of ethanolamine and methanol at 65 °C for 30 min [4]. We have found that the first method is not suitable for deprotecting arrays made on glass derivatised using the ‘vapour-phase’ method (see Sect. 3.1.1). 3.2 Hybridisation of a Labeled Target to Scanning Arrays

The target nucleic acid is labeled with a radioisotope (33P or 32P) or a fluorescent compound (e.g., CY5) and hybridised to a scanning array. The choice of label depends on the application. For example, when studying folding of a target nucleic acid, it may be more appropriate to label it with a radioisotope rather than CY5 because the bulky CY5 groups may interfere with or alter the folding of the labeled compound. The labeled product is generated by the standard in vitro transcription in the presence of a labeled precursor (such as [α-33P]UTP or [α-32P]UTP or CY5UTP), using an appropriate DNA template. In vitro transcription is generally carried out using T7, T3 or SP6 RNA polymerase. A plasmid containing the desired fragment under the transcriptional control of a bacteriophage promoter (Table 2) can be used as a template. The plasmid is linearised with an appropriate restriction endonuclease to produce transcripts of a defined length without contaminating vector sequence. If the fragment of interest has not been cloned into such a plasmid, the template for use with a bacteriophage RNA polymerase can also be generated using a polymerase chain reaction (PCR): primers used in the PCR reaction are such that one of them has a bacteriophage promoter/leader sequence added at the 5′-end (Table 2). Approximately 50–70 fmol of labeled transcript are used in hybridisation. The transcript is diluted in an appropriate volume of the hybridisation buffer. For arrays made on glass, the hybridisation solution is applied as a thin film of liquid between the array and a glass plate of the same size. Polypropylene arrays are hybridised in a rotating glass tube placed in a hybridisation oven at an appropriate temperature. Arrays made on both glass and polypropylene can be stripped of target and reused several times.We have used the arrays made on polypropyene in as many as 7–8 hybridisations without significant loss of signal, after which they tend to deteriorate very quickly. Stripping solution is 100 mM sodium carbonate/bicarbonate buffer at pH 9.8–10 which is heated to ~90 °C and the arrays is dipped into it for 2–3 min. The arrays are stored in absolute ethanol at 4 °C. Table 2. Bacteriophage promoter and leader sequences a

T7 (or) T3 SP6 a

5′ taa tac gac tca cta ta ggg cga 5′ taa tac gac tca cta ta ggg aga 5′aat taa ccc tca cta aa ggg aga 5′ att tag gtg aca cta ta gaa tac

The hexa-nucleotide leader sequence (in italics) appears in the transcript.

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3.3 Reading a Scanning Array Image

After hybridisation with a radiolabeled target, exposure to a storage phosphor screen captures the hybridisation signal from the array. The screen is then scanned on a PhosphorImager (such as the STORM; Molecular Dynamics). For a fluorescently labeled target, the array is scanned directly on a PhosphorImager. Visual inspection of the image reveals the results generally, but computer-assisted analysis is needed to extract quantitative information about the hybridisation capacities of the various oligonucleotides and their exact sequence and length. We use a software package called xvseq to analyse scanning array images that is run on a SUN Solaris workstation (available by anonymous ftp at (ftp://bioch.ox.ac.uk/pub/xseq.tar.gz)). The programme reads and displays im-

Fig. 3. Display and analysis of a scanning array image using xvseq. A The results of the inte-

grated pixel values are displayed as paired histograms and the two values are for the halves above and below the centre line of the array. Only values for 20-mers are displayed here but the programme can display all values from monomers to the longest in the set. The programme also has several other useful features. For example, clicking on a cell in the array highlights the corresponding region in the sequence, the relevant bar in the histogram and the integrated pixel values of the two cells. Clicking on a bar in the histogram (filled bar) highlights the corresponding cells of the array and the sequence (boxed sequence). B Image of a scanning array obtained after hybridisation of a 32P-labeled transcript. C A part of the image magnified with a template grid overlay

Oligonucleotide Scanning Arrays

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ages generated by a Molecular Dynamics PhosphorImager and can perform standard image manipulations such as scaling, clipping, rotation and colormap control. Its main purpose, however, is to calculate and display integrated intensities of array oligonucleotides. The programme generates a template grid of overlapping diamonds (or circles) that is superimposed on the array image, and allows the user to specify the template size, shape (circular or diamond-shaped), location, step size between successive templates, as well as the sequence that generated the array pattern. The template grid allows accurate registration of the oligonucleotides on the array.An example of analysis of an image using this program is given in Fig. 3.

4 Applications 4.1 High-Throughput Screening of Antisense Reagents

Antisense reagents comprise two major classes, antisense oligonucleotides and ribozymes. Antisense oligonucleotiodes act by binding to the target mRNA and mediating its cleavage at the heteroduplexed site by an RNAse H-based mechanism, or by strongly binding to the target site and thus interfering with its translation. Ribozymes are catalytic RNA molecules that are capable of mediating cutting at the target sequence. Such reagents can therefore be used, in principle, to explore function of a gene, and are potential therapeutics for many diseases caused by abnormal or overexpression of genes, such as those involved in cancers or those of viral origin. Both types of antisense reagents require a site on the target mRNAs where they can bind efficiently. Finding such sites is not an easy task; it is generally found that only 5%–10% of the chosen sites allow efficient binding by the antisense reagents and, in addition, antisense reagents placed only a single or a few bases apart can have quite different antisense activities. There is now convincing evidence that intramolecular folding in RNAs renders most of the molecule inaccessible to intermolecular binding [5–10]. However, folding of RNAs is difficult to predict [11] and the rules that govern heteroduplex formation are poorly understood [12]. Effective antisense reagents are thus found empirically, and a large number of reagents need to be individually synthesised and screened for their binding capacities with the target sequence.A full review of the screening methods to find effective antisense reagents is beyond the scope of this article and is presented elsewhere (e.g., [13–15]). The scanning arrays provide an efficient method to screen for open structures in RNAs that can be targeted with antisense reagents and allow a large number of reagents to be screened simultaneously in simple hybridisation tests. This method provides quantitative measurements of the relative accessibility of every site in a RNA and thus facilitates selection of the optimal target sequence for designing antisense reagents. Hybridisation to scanning arrays is typically carried out in 1 M NaCl, which is far from being physiological. However, we have found an excellent correlation between the binding shown by oligonucleotides on arrays under these conditions and their antisense activities in many systems [8, 16,

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17). Milner et al. [8] used β-globin mRNA as target and showed that duplex yield on a scanning array of anti-β-globin antisense oligonucleotides correlated with their inhibitory potential in an in vitro translation assay. We have also tested the antisense activities of over 30 oligonucleotides targeted to mRNAs of cyclins B1, B4 and B5 of Xenopus laevis in cell-free egg extracts and oocytes [17]. Scanning arrays corresponding to the first 120 nucleotides of each of the three B-type cyclins were hybridised to their corresponding transcript. Only some of the oligonucleotides were found to produce strong heterduplexes and in many cases a single or a few bases shift produced significant changes in the heteroduplex yields. Oligonucleotides that produced strong heteroduplex yield, and others that showed little annealing, were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between the antisense potency and the affinities of the oligonucleotides for cyclin transcripts as measured by the arrays, despite the complexity of the cellular environment.Although a role for cellular proteins and other small molecules in influencing the binding of antisense reagents to their targets has been proposed [18, 19], surprisingly our results suggest that it may not be a major factor. 4.2 The Study of Nucleic Acid Folding and Heteroduplex Formation

In order to develop theoretical methods to predict intermolecular binding, one needs to understand not only the folding process of nucleic acids, but also the mechanism of duplex formation. Scanning arrays have proved to be useful tools to study both these aspects. 4.2.1 RNA Folding

Folding pathways of large RNAs (as large as mRNAs) are poorly understood. There are two generally held views: one is that most secondary interactions are local and are established as RNA is transcribed; the alternative view is that RNAs (including mRNAs) exist in a global minimum free energy state – a state that would have to be established after the synthesis was complete. However, there is little evidence to support either view. We used a scanning array to explore these suggestions. With compelling evidence that intermolecular binding largely depends on folding of the interacting species [5–10], we assumed that the interaction of short oligonucleotides with an RNA could be used as a tool to study changes in the folding of an RNA molecule [20]; we speculated that a change in the secondary structure of an RNA would also result in alteration of its interaction with complementary oligonucleotides – accessible regions may become inaccessible, and vice versa. To mimic the synthetic pathway, we made transcripts that had a common start point (with one exception) but widely different end points by transcribing restriction fragments with different lengths; the other had a small deletion at the 5′-end. These transcripts were hybridised to an array that had oligonucleotide

Oligonucleotide Scanning Arrays

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complementary to a part of the common sequence shared by the transcripts. We expected that, if these transcripts were to reach a global free energy minimum, the transcripts with different sizes would acquire different secondary structures and so produce different hybridisation patterns. Theoretical free energy calculations of the transcripts did predict quite different secondary structures. Strikingly, the predominant features of the hybridisation pattern were shared by all the transcripts. These data suggested that RNA folding creates local stable states that are trapped early in the transcription or folding process and that these states are not altered to produce structures with global minimum free energy. 4.2.2 Heteroduplex Formation

For two main reasons it is difficult to predict the sites on a RNA that would hybridise with antisense reagents. First, despite the elegance of the computational methods, there is a great deal of uncertainty in the prediction of folding in mRNAs. Second, the rules that govern intermolecular binding are also poorly understood. If predictive approaches are to be useful, both these aspects need to be fully understood. Mir and Southern [12] studied the mechanisms of heteroduplex formation by studying the binding of oligonucleotides to structurally well-characterised tRNAphe in solution. In an exhaustive assay they used an array of 12-mer (and shorter) oligonucleotides complementary to tRNAphe to measure binding to radiolabeled tRNAphe. The hybridisation data were analysed on the basis of the clover-leaf secondary and three-dimensional crystal structures of tRNAphe. They derived certain rules from this study. They found that most of the tRNA was inaccessible and as small as a single base shift produced significant changes in the hybridisation yields. All regions of tRNA that formed heteroduplexes had unpaired bases stacked onto the end of a stem and most of the bases that were incorporated into the heteroduplex were part of a continuous helix within the tRNA and on one side of the stem. Strikingly, there were several other oligonucleotides with similar features and overlapping sequences that did not produce heteroduplexes with tRNA. The oligonucleotides that gave the highest heteroduplex yields were accommodated within the existing higher-order stacking architecture of the native structure. They also showed that binding of oligonucleotides could be affected by oligonucleotide length as well as sequence. Overall, the factors that influenced the binding were embedded in the structure of tRNA. 4.2.3 A Study of the Secondary Structure of Nucleic Acids with Modified Bases

Nguyen and Southern [21] used scanning arrays to study the hybridisation of a target DNA containing modified bases. The aim of this investigation was to find ways to destabilise the secondary structure in the target that has such a strong influence on intermolecular binding. As a model, they used a DNA target with a self-complementary sequence forming a stem loop. They replaced dC with N-4ethyldeoxycytidine (d4EtC) that specifically hybridises with natural dG to give a

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G: 4EtC base pair but with reduced stability compared with the normal G:C base pair. By hybridising it to an array of complementary oligonucleotides, they found that this substitution greatly weakened the target’s secondary structure and allowed it to hybridise to the complementary oligonucleotides more strongly than with the natural analogue.

5 Concluding Remarks Scanning arrays are versatile tools for the analysis of nucleic acid interactions. These can therefore provide a platform to improve the array-based methods. The applications of scanning arrays discussed in this article are only those that have been published to date. Undoubtedly, new applications of this method will emerge in the future and combination of scanning arrays with conventional approaches may provide a more powerful way forward in the study of nucleic acid interactions. Acknowledgements. We would like to thank all past and present members of the group who con-

tributed to the development of scanning arrays and Martin Johnson for his description of mask construction. The Medical Research Council, UK, funded M. Sohail.

6 References 1. Maskos U, Southern EM (1992) Nucleic Acids Res 20:1679 2. Matson RS, Rampal JB, Coassin PJ (1994) Anal Biochem 217:306 3. Sohail M, Southern EM (2001) In: Rampal JB (ed) Methods in molecular biology: oligonucleotide arrays – methods and protocols, vol 170. Humana Press, pp 181–199 4. Polushin NN, Morocho AM, Chen B-C, Cohen JS (1994) Nucleic Acids Res 22:639 5. Lima WF, Monia BP, Ecker DJ, Frier SM (1992) Biochemistry 31:12055 6. Southern EM, Case-Green SC, Elder JK, Johnson M, Mir KU, Wang L, Williams JC (1994) Nucleic Acids Res 22:1368 7. Bruice TW, Lima WF (1997) Biochemistry 36:5004 8. Milner N, Mir KU, Southern EM (1997) Nat Biotechnol 15:537 9. Patzel V, Sczakiel G (1998) Nat Biotechnol 16:64 10. Sohail M, Akhtar S, Southern EM (1999) RNA 5:646 11. Michel F, Westhof E (1990) J Mol Biol 216:585 12. Mir KU, Southern EM (1999) Nat Biotechnol 17:788 13. Branch A (1998) Antisense Nucleic Acid Drug Dev 8:249 14. Sohail M, Southern EM (2000) Curr Opin Mol Ther 3:264 15. Sohail M, Southern EM (2000) Adv Drug Deliver Rev 44:23 16. Southern EM, Milner N, Mir KU (1997) In: Chadwick DJ, Cardew G (eds) Ciba Foundation Symposium 209, John Wiley, Chichester, p 38 17. Sohail M, Hochegger H, Klotzbucher A, Guellec RL, Hunt T, Southern EM (2001) Nucleic Acids Res 29:2041 18. Bertrand EL, Rossi JJ (1994) EMBO J 13:2904 19. Herschlag D, Khosla M, Tsuchihashi Z, Karpel RL (1994) EMBO J 13:2913 20. Zarrinkar PP, Williamson JR (1994) Science 265:918 21. Nguyen H-K, Southern EM (2000) Nucleic Acids Res 28:3904 Received: June 2001

CHAPTER 4

The Use of MassARRAY Technology for High Throughput Genotyping Christian Jurinke · Dirk van den Boom · Charles R. Cantor · Hubert Köster SEQUENOM, Inc., 3595 John Hopkins Court, San Diego, CA 92121,USA. E-mail: [email protected]

This chapter will explore the role of mass spectrometry (MS) as a detection method for genotyping applications and will illustrate how MS evolved from an expert-user-technology to a routine laboratory method in biological sciences. The main focus will be time-of-flight (TOF) based devices and their use for analyzing single-nucleotide-polymorphisms (SNPs, pronounced snips). The first section will describe the evolution of the use of MS in the field of bioanalytical sciences and the protocols used during the early days of bioanalytical MALDI TOF mass spectrometry. The second section will provide an overview on intraspecies sequence diversity and the nature and importance of SNPs for the genomic sciences. This is followed by an exploration of the special and advantageous features of mass spectrometry as the key technology in modern bioanalytical sciences in the third chapter. Finally, the fourth section will describe the MassARRAY technology as an advanced system for automated high-throughput analysis of SNPs. Keywords: SNP, High-throughput, MALDI mass spectrometry, Genomics, DNA chip

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Mass Spectrometry . . . . . . . . . . . . . . . ESI MS . . . . . . . . . . . . . . . . . . . . . . MALDI-TOF MS . . . . . . . . . . . . . . . . Mass Spectrometry in the Protein World . . . Mass Spectrometry in the Carbohydrate World Mass Spectrometry in the DNA World . . . . .

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Intraspecies Sequence Variations . . . . . . . . . . . . . . . . . Restriction Fragment Length Polymorphisms and Short Tandem Repeats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Single Nucleotide Polymorphisms . . . . . . . . . . . . . . . . . Pharmacogenomics . . . . . . . . . . . . . . . . . . . . . . . . .

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List of Symbols and Abbreviations ESI LCR MALDI MS PCR PSD RFLP SNP STR TOF VNTR

Electrospray ionization Ligase chain reaction Matrix assisted laser desorption/ionization Mass spectrometry Polymerase chain reaction Post source decay Restriction fragment length polymorphism Single nucleotide Polymorphism Short tandem repeat Time-of-flight Variable number tandem repeats

1 Mass Spectrometry in the Biological Sciences 1.1 Mass Spectrometry

The general principle of mass spectrometry (MS) is to produce, separate and detect gas phase ions. Traditionally, thermal vaporization methods are used to transfer molecules into the gas phase. The classical methods for ionization are electron impact (EI) and chemical ionization (CI). Most biomolecules, however, undergo severe decomposition and fragmentation under the conditions of both methods. Consequently, the capabilities of mass spectrometry have been limited to molecules the size of dinucleotides [1]. Analysis of oligonucleotides with a mass range of up to 3000 Da became feasible with the development of plasma desorption (PD) methods [2]. However, until the invention of ‘soft’ ionization techniques such as ESI- and MALDI MS, mass spectrometric tools were not widely considered for routine applications in biological sciences. 1.2 ESI MS

Electrospray ionization (ESI) was first introduced by Fenn et al. in the late 1980s [3]. The method is based on spraying an analyte through a syringe into an electrical field. Ionization of analyte molecules consists of coulomb explosions, resulting in fully desolvated ions migrating towards the mass analyzer. The spectra obtained by ESI MS are mostly composed of multiple charged ions of the

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parent molecules. The analyte mass is determined by deconvolution of the complex spectra. Application of ESI MS to the analysis of nucleic acids has been described by several authors [4, 5]. However, today the most common bioanalytical applications of ESI MS are in the field of peptide and protein analysis [6, 7]. The main reasons for this may be the higher duty cycle of MALDI MS, an important factor for routine high-throughput applications, and the complex signal patterns obtained from ESI MS combined with a comparably low tolerance for impurities. 1.3 MALDI-TOF MS

Matrix assisted laser desorption/ionization (MALDI) as a principle for analysis of large biomolecules was introduced by Karas and Hillenkamp in 1989 [8]. Briefly, in MALDI MS, the sample is embedded in the crystalline structure of small organic compounds (matrix) and deposited on a conductive sample support. The co-crystals are irradiated with a nano-second laser beam, for example a UV laser with a wavelength of 266 nm or 337 nm. The energies introduced are in the range of 1¥107 to 5¥107 W/cm2. The laser energy causes structural decomposition of the irradiated crystal and generates a particle cloud (the plume) from which ions are extracted by an electric field. The mechanism behind this not fully understood process of desorption may be best described as a conversion of laser energy to vibrational oscillation of the crystal molecules which finally results in the disintegration of the crystal.After acceleration, the ions drift through a field-free path (usually one to two meters in length) and finally reach the detector (e.g. a secondary electron multiplier or channel plate) (see Fig. 1). Ion masses (mass-to-charge ratios, m/z) are typically calculated by measuring their time-of-flight (TOF) which is longer for larger molecules than for smaller ones (provided their initial energies are identical). Since single charged, non-fragmented ions are predominantly generated, parent ion masses can easily be determined from the resulting spectrum without the need for complex data processing. The masses are accessible as numerical data for direct processing and subsequent analysis. TOFs measured during a typical MALDI experiment are in the range of several microseconds. The quality of the spectra, which is reflected in terms of resolution, mass accuracy, signal-to-noise ratio, and also sensitivity, is highly dependent on sample preparation and on the choice of matrix compounds [9]. The predominant effects influenced by sample purification and matrix choice are adduct formation and fragmentation. Both will be described in detail in the following sections. In brief, the purification process should result in a sample which is free of crystallization disturbing agents (like detergents, urea or DMSO), adduct forming agents (such as unvolatile cations) and not too diluted. The classical method of preparing a sample on a holder, still widely used today, is the so called ‘dried-droplet’ method [10]. It relies simply on pipetting a small volume of the sample (usually about 0.5 ml) to a drop (about 1 ml) of matrix solution and allowing the mixture to dry. For high throughput applications, as described later, this procedure has to be modified.

Fig. 1. Schematic representation of MALDI-TOF mass spectrometer

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1.4 Mass Spectrometry in the Protein World

Both, ESI and MALDI TOF MS have been proven as valuable research tools for the analysis of peptides and proteins and also for the investigation of their interactions with each other or different substances. The characterization of proteins was one of the first described applications of MALDI MS. Proteins and peptides are easier to volatilize than nucleic acids. Besides analyzing peptide patterns from cell lysates or serum, recent interesting applications include the analysis of patterns from differentiated tissues of organs and the typing of bacteria using whole cells. An exciting demonstration of the analytical power of MALDI MS is the investigation of the neurotransmitter content of single neurons [11]. An advantage of MALDI compared to ESI is that MALDI can deal with significantly higher amounts of impurities. For example, it is possible to analyze hemoglobin proteins directly from blood [12]. The analysis of milk proteins without any purification has also been demonstrated [13]. Caprioli and coworkers described a direct profiling of proteins in tissue sections [14]. The authors were able to distinguish between proximal, intermediate, and distal colon from fresh tissue blotted on a conductive polyethylene membrane. Prior to this work, Eckerskorn et al. demonstrated that MALDI analysis of blotted proteins after gel electrophoresis is feasible [15]. Protein- and peptide conjugates are another field of application where MS techniques provided valuable contributions. An example is the analysis of antibody conjugates by MALDI TOF [16]. Furthermore, an emerging field is the analysis of non-covalent protein-DNA complexes and interactions [17, 18]. 1.5 Mass Spectrometry in the Carbohydrate World

Another area of bioanalytics where MALDI TOF plays an important role is composition analysis of complex carbohydrates or glyco-conjugates such as glycopetides or glycoproteins. A very basic application is the analysis of complex carbohydrate solutions [19]. A more sophisticated application is the compositional analysis of carbohydrate components of complex proteins, for example elucidation of glycosylation sites [20] and composition analysis of carbohydrate side chains. For this purpose, enzymatic methods are combined with a special feature of MALDI, post source decay [21].As described in Sect. 1.6, fragmentation of analyte molecules can occur throughout the MALDI process, even if the desorption and ionization is comparably soft. If this fragmentation occurs after the ions have left the source area of the mass spectrometer, it is referred to as post-source-decay (PSD). Special instrumentation (ion gates) are used to allow only ions of defined flight-time to proceed towards the detector. With this method, fragments deriving from a certain parent ion can be analyzed. Reflectron instruments have to be employed to resolve and detect the ion subspecies. Using this instrumentation, oligosaccharide mixtures cleaved from glycopeptides by treatment with enzymes like PNGase can be analyzed. By careful interpretation (using spectra libraries for comparison) the structural composition of the glycan side chains can be revealed [22].

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1.6 Mass Spectrometry in the DNA World

The application of MALDI MS to the analysis of nucleic acids has for years remained a field of small impact for the bio-science community. Nucleic acids are far more difficult to analyze under MALDI conditions than peptides, so applications have been limited. The reason is that nucleic acids are especially susceptible to adduct formation and fragmentation. Because of their negatively charged phosphate backbone, nucleic acids are polyanions.As such, they tend to form salt adducts with cations present in the surrounding medium. In biochemical reactions, these are predominantly sodium and potassium ions. The result of adduct formation is a distribution of the signal: a main signal with the protonated analyte may be accompanied by signals resulting from multiple adduct formation. For example, every sodium ion attached to the analyte molecule will cause an additional signal with a mass of plus 23 Da. Consequently, adduct formation lowers sensitivity and analytical accuracy – the total amount of ions is distributed over a multitude of ion species, and the mass differences between those may become too small to be resolved.Approaches to overcome adduct formation are purification and ion-exchange procedures based either on solid-phase [23], or cation exchange resin methods [24]. The chemical modification of the phosphate backbone has also been proposed for preventing adduct formation [25]. The addition of ammonium containing additives such as di-ammonium citrate [26] or tartrate [27] to the matrix has a huge impact on cation heterogeneity of analytes. The use of mM solutions of ammonium hydroxide during the conditioning process has also a beneficial effect [28]. The reason for exchanging cations for ammonium is that the latter is a volatile cation which is released as ammonia in the gas phase leaving the analyte molecules as free acids. Based on the chemical nature of nucleic acids, some fragmentation reactions can occur relatively efficiently during the MALDI process. The predominant fragmentation is depurination. Protonization of the nucleobases A and G (at position N7) induces a polarization of the N-glycosidic bond between sugar and nucleobase finally resulting in nucleobase elimination. Subsequently to depurination further fragmentation can occur via backbone cleavage.Also the loss of C has been reported [29] which is mediated through protonation at N3. It has been demonstrated by Hillenkamp and coworkers that RNA is comparably more stable under MALDI conditions than DNA. The proposed reason is the lack of the 2¢ hydroxy group in the ribose sugar moiety [30]. Approaches to compensate for the detrimental influence of adduct formation have been contributed by instrument developers and molecular biologists. It has been demonstrated by several groups that introduction of 7-deaza nucleobases, where N7 is replaced by C, is helpful in suppressing depurination reactions [31, 32], since the proton acceptor site is removed. An important contribution to analyzing DNA with MALDI-TOF is also the introduction of delayed extraction instruments by Wiley and McLaren [33]. Compared to static extraction, which employs a permanent electrical field for ion acceleration, the delayed extraction accumulates ions over a time range of some

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nanoseconds before the extraction voltage is applied. The effect is a compensation of the initial differences in kinetic energies of the analyte molecules, since molecules with higher initial velocities expand further during the delay time and experience a lower potential during acceleration. To some extent, this may also compensate for positional variations of molecules which occur due to an inhomogeneous distribution in the matrix cocrystals. The introduction of favorable matrixes [34] for DNA analysis has led to the development of many different applications ranging from oligonucleotide sequence analysis [35, 36] and PCR- or LCR product detection [37, 38] to PCR product sequencing [39], mutation detection [40, 41] and clinical diagnostics [42, 43]. Furthermore, strategies to analyze combined amplification and sequencing reactions have been described [44] and the analysis of in vitro transcripts has also been reported [45]. On the more analytical side, fragmentation mechanisms for nucleic acids under MALDI conditions [46] and the conditions for the analysis of intact double-stranded DNA have been investigated [47]. Some of the latest and most promising lines of development are the application of IR-MALDI for the analysis of DNA molecules of up to 2.1 kb by Hillenkamp and coworkers [48] and the description of bench-top MALDI instruments for biopolymer analysis by Cotter and coworkers [49].

2 Polymorphic Markers in the Genomic Sciences 2.1 Intraspecies Sequence Variations

Within the foreseeable future, the efforts of the Human Genome Project (HGP) will lead to a reference sequence for Homo sapiens. A ‘working version’ of the human genome sequence – covering about 97% of the entire genome – was presented to the public on the 26 of June 2000, jointly from scientists of the HGP and Celera. Today, the research organizations associated in the HGP produce about 1000 bases of raw sequence data per second – 24 hours a day and seven days a week. With this dedicated effort, we can expect the remaining gaps to be closed by 2003 or earlier. However, our knowledge of the architecture and function of the human genome is poor – there are still disputes on whether there are 40,000 or 140,000 genes in the human genome [50] and on the role of the so called ‘junk DNA’. Another blind spot on our map is the encoding of intraspecies genetic diversity.We estimate that the genetic diversity within the human species is about 0.1 to 0.3%. For purpose of comparison, the genetic identity between Homo sapiens and both Pan species (Chimpanzees and Bonobos) is about 98.4% – which is closer than the relationship between African and Indian elephants. The knowledge of the 3.2 billion base pair human sequence information and information on our genetic diversity are milestones in our understanding of the contribution of genes to many complex problems such as disease predisposition and diversity of drug response by different patients. The manifestations of genetic diversity within the human genome are mostly SNPs, RFLPs (which can be

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treated somehow as a subset of SNPs), STRs and random mutations. The nature and importance of these polymorphic genetic elements are discussed in the following sections. 2.2 Restriction Fragment Length Polymorphisms and Short Tandem Repeats

RFLPs (restriction fragment length polymorphisms) can be defined as (single) nucleotide polymorphisms located in the recognition or cleavage site of a restriction endonuclease, thus altering the corresponding cleavage pattern. With this definition, RFLPs are bi-allelic markers with the allele status permissive to cleavage, yes or no.Although RFLP markers [51] are useful in many applications, they are often of poor information content, for example in family linkage studies and their analysis is cumbersome to automate. STR markers (short tandem repeats, microsatellites) [52], in contrast, are quite highly informative (through their polymorphic number of repeats) and easy to prepare and analyze using PCR-based assays with a considerable potential for automation. However, with conventional gel electrophoresis-based analysis, typing of large numbers of individuals for hundreds of STR markers still remains a challenging task. Today, the application of STR analysis is mainly in the field of identification (forensics) and agricultural breeding approaches, covering both, plants and animals. The development of markers to characterize intraspecies sequence variations has, from a more theoretical point of view, evolved from the use of RFLPs to microsatellites and very recently to SNPs. 2.3 Single Nucleotide Polymorphisms

Single nucleotide polymorphisms (SNPs) can be defined as bi-allelic base variants within a population occurring with an allelic frequency higher than 1% (with 1% as a more or less arbitrary threshold). SNPs can be seen as a generalization on single nucleotide polymorphisms, covering also RFLPs. The importance of SNPs to our understanding of genetic diversity is recognized today by many academic and commercial organizations. Consequently this is leading to huge SNP discovery efforts within the human genome. The program which has received the most public attention is The SNP Consortium (TSC), a collaboration between 13 multinational companies and prominent academic institutions like the Washington University, the Whitehead Institute and The Sanger Centre. The declared goal of TSC is to deliver 300,000 SNPs to the public domain by the end of 2001. Currently, more than 100,000 SNP have been disclosed to the public [53]. The Human Genome Project (HGP) also has an ongoing human diversity project, which will lead to a genome-wide map of 100,000 SNPs [54]. The strategies for discovering SNPs are either direct or indirect approaches. For a direct approach, certain regions of the genome, e.g. genes which may be interesting disease predisposition candidates, are chosen and screened thoroughly

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for all SNPs occurring in many different individuals. This kind of study is referred to as a high saturation study and will lead to a defined set of SNPs. Since the target genes are pre-selected, there is a high probability of identifying a diseasecausing SNP by selecting those polymorphisms which alter amino acid (codingor cSNPs) or regulatory sequences for subsequent association studies. Another, more indirect and broad approach is based on mining polymorphisms from existing data bases by comparing overlapping sequences. This approach results in so called in silico SNPs (isSNPs). The isSNPs need to be validated or confirmed since they can result either from sequencing errors or real polymorphisms. For the validation of isSNPS, real DNA samples need to be analyzed. The technologies used to discover SNPs are diverse and include re-sequencing (either gel- or microchip hybridization based), SSCP, HPLC and enzymatic cleavage methods [55, 56]. In whatever manner they have been discovered, SNPs need to be analyzed among many different populations to reveal their meaning for human healthcare. A direct way to benefit from information about human diversity is to compare the allelic frequencies of SNPs in populations of individuals, either affected or unaffected by a certain disease. Candidate genes, supposed to be causative for the disease of interest are carefully selected by analyzing metabolic pathways which are involved in the etiology of the particular disease. Such a study results in the association of certain SNPs with the manifestation of a clinical status. This approach as for example resulted in knowledge about susceptibility to HIV infection, which has been demonstrated [57] to correlate to the genetic status of a chemokine receptor (CCR5). Another example is the correlation of deep-vein thrombosis to a polymorphic variation in coagulation factor gene FV [58]. A more indirect way to capitalize from knowledge about human diversity is to analyze the reasons for differences in drug metabolism; this approach is called pharmacogenomics. 2.4 Pharmacogenomics

Traits within populations, such as the ABO blood groups, are phenotypic expressions of genetic diversity. This is also the case for certain variations in response to drug therapy.When taken by poor metabolizers, some drugs may cause exaggerated pharmacological response and adverse drug reactions. For example, tricyclic antidepressants exhibit order-of-magnitude differences in blood concentrations depending on the status of patients’ metabolizing enzymes [59]. Pharmacogenomics and pharmacogenetics are often used as synonyms. However, pharmacogenetics is the study of genetic polymorphism in drug metabolism and pharmacogenomics is more an umbrella term for the optimization of drug discovery and development using genetic knowledge. The major idea behind the efforts of such development is to provide people with individualized drugs. Instead of having one block buster drug which should fit for all patients and ignoring adverse drug reactions, pharmaceutical companies are now thinking about providing drugs especially for certain populations. For example, pharmaceutical companies can screen individuals for specific genetic polymorphisms

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before entry into clinical trials to ensure that the study population is both relevant and representative. Targets for such screenings are cytochrome P450 enzymes or N-acetyltransferase isoenzymes (NAT1 and 2). Potential drug candidates affected by polymorphic metabolism include antidepressants, antipsychotics and cardiovascular drugs. Another possible application deriving from knowledge of genetic diversity is the potential of rescuing drugs for the market. A promising drug candidate, which fails to get approval from regulatory authorities because of certain side-effects may enter the market with a restricted approval for application on patients with a well-defined genetic background.At first glance, this may sound like a softening of the restrictive approval procedures. However, by taking into account that these drugs will only be prescribed after careful examination of the individual patient, this concept opens the way to bring highly efficient drugs to people, with the knowledge that unwanted side effects will be excluded. Since the money spent on the development of failed drugs finally is amortized through approved drugs, a positive side effect of this strategy may be that drug treatment, over all, will become cheaper. Of course, the methods employed for such approaches need to fulfill strict quality requirements, since the outcome of an error is potentially fatal.

3 The Advantages of Mass Spectrometry for Genetic Diversity Detection The requirements for SNP discovery- and SNP typing (or scoring) technologies are quite different. All of the above-mentioned methods for SNP discovery have merits. For large scale SNP scoring projects, however, they will fail to be efficient. The reasons are a lack of reasonable automation and an inherent susceptibility to errors. The first issue is mainly due to gel electrophoresis technology which is cumbersome to automate.Another issue is caused by indirect detection methods based on labels. The inherent and clearly predominant advantage of mass spectrometry as a detection device is a direct detection of the analyte itself. This is a paradigm shift for molecular biology, since it eliminates all uncertainties caused by indirect detection via labels. This may be illustrated by an example. Consider a certain PCR amplicon which is subject to unbalanced PCR (one allele is amplified with significant preference). Consider further, that mismatches can occur if hybridization is used to identify the alleles of a SNP. This generates some background signal. With these two inherent processes in mind, one can easily imagine that there is a point where the signal intensities generated by either effect can no longer be distinguished. That means a true heterozygote affected by imbalanced PCR generates the same signal as a mismatched probe. This effect needs to be compensated by a redundant experiment setup and careful statistical analysis. From pure analytical and also practical points of view these are drawbacks which can be overcome, if MS is applied. The dynamic range of analyzing MassEXTEND reactions with MALDI TOF allows for the detection of allele ratios of up to 1:50. The analytical accuracy of MALDI TOF is about 0.1 to 0.01% of the determined mass. This means a 15mer DNA with about 4500 Da can unambiguously be distinguished from a 16mer which is approximately 300 Da, or 6.6%, higher in mass.

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A major factor which needs to be taken into account is the cost of a whole genotyping project – not just the cost of individual SNP typing reactions. A major factor in these costs of course is data analysis by highly trained specialists. Furthermore, every new SNP assay needs a considerable time for implementation using standard curves and statistics. These are all certain drawbacks in a high throughput environment. Assay design for MassEXTEND reactions can be done automatically and implementation steps as necessary for fluorescence-based assays are obsolete. An important feature in the cost per assay is the capability of easy multiplexing. Multiplexed assays are conducted together in one tube (or one well of a micro plate) and combined; for example, assays are combined for different SNPs to save time and reagents. With fluorescent detection, the level of multiplexing is somewhat coupled to the amount of available dyes – which is in most applications only four. For MALDI-based SNP detection, a multiplex level of up to 12 assays has already been described [60]. These advantages taken together may be the reason that mass spectrometric based methods have been referred to as the ‘gold standard’ for SNP analysis in a recent review on this topic [61].

4 MassARRAY and High-Throughput Genotyping The MassEXTEND reaction was developed by Sequenom especially for the purpose of assessing genetic polymorphism by mass spectrometry. The assay format can be used for the analysis of deletion-, insertion-, and point-mutations, STR-, and SNP-analysis, and it allows for the detection of compound heterozygotes. The MassEXTEND process comprises a post-PCR primer extension reaction carried out in the presence of one or more dideoxynucleotides (ddNTPs) and generates allele-specific terminated extension fragments (see Fig. 2). For SNP analysis the primer binding site is placed adjacent to the polymorphic position. Depending on the nucleotide status of the SNP, a shorter or a longer extension product is generated. In the case of heterozygosity, both products are generated.After completion of the reaction, the products are purified and analyzed by MALDI TOF MS [62]. In the example given in Fig. 2, both elongation products are expected to differ in mass by one nucleotide. Raw data for a heterozygous DNA sample analyzed by a MassEXTEND assay are given in Fig. 3. The two SNP alleles appear as two distinct mass signals. Careful assay design makes a high-level multiplexing of MassEXTEND reactions possible. In the case of STR analysis, a ddNTP composition is chosen which terminates the polymerase extension at the first nucleotide not present within the repeat [63). For length determination of a CA repeat a ddG or ddT termination mix is used. Even imperfect repeats harboring insertion or deletion mutations can be analyzed with this approach. Figure 4 displays raw data from the analysis of a human STR marker in a heterozygous DNA sample. Both alleles differ by 4 CA repeats. The DNA polymerase slippage during amplification generates a pattern of ‘stutter fragments’ (marked with in asterisk in Fig. 4). In case of heterozygotes

Fig. 2. Reaction scheme for MassEXTEND reaction

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Fig. 3. Raw data for a typical MassEXTEND reaction

which differ in just one repeat, the smaller allele has higher intensities than the larger allele, because allelic and stutter signals are added together. When compared to the analysis of hybridization events by detecting labels – even on arrays, the DNA MassARRAY approach differs significantly. The MassEXTEND assay is designed to give only the relevant information. The mass spectrometric approach enables a direct analyte detection with 100% specificity and needs no redundancy. This accuracy and efficacy is combined with sample miniaturization, bioinformatics and chip-based technologies for parallel processing of numerous samples . The use of an advanced nano-liquid handling combined with surface modified silicon chips permit today the automated scanning of 384 samples in about

Fig 4. Raw data of MassEXTEND reaction for STR analysis

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Fig. 5. SpectroCHIP with 384 different samples

40 minutes. From the mass spectrometric point, the advantage of this sample preparation method is the generation of extremely homogeneous crystals allowing for an automated scanning with just ten laser shots per sample. Currently up to 10 SpectroCHIPs each containing up to 384 different samples (see Fig. 5) can be analyzed in one unattended run using a Bruker/SEQUENOM SpectroREAD mass spectrometer. The SpectroREAD addresses each position of the chip sequentially, collects the sum of ten laser shots, processes and stores the data and proceeds to the next spot of the chip. Using a proprietary algorithm, masses as well as signal-to-noise ratios are automatically analyzed and interpreted. After completion of analysis, the results are transferred to a data base and stored as accessible genetic information (see Fig. 6). The database also provides a tool for visual control and comparison of spectra with theoretically expected results. MassEXTEND assays can easily be designed in a high throughput mode with a computer aided assay development module. A very promising tool in combination with MassARRAY is the analysis of allelic frequencies by means of pooled DNA samples. In this approach a significant number of different genomic DNA samples (e.g. 100 individual DNAs) are combined in equimolar amounts to generate a DNA pool. This pooled population is subjected to PCR amplification and analyzed in a single MassEXTEND reaction. If carefully analyzed, the outcome of such an experiment is information about the

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Fig. 6. Screenshot of processed mass spectrum with genotype analysis

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frequencies of the individual alleles of a SNP. The information generated is extremely useful in several ways. It can be used to validate a SNP – to decipher whether an isSNP is really a relevant polymorphism rather than a sequencing artifact. The allelic frequency can, furthermore, be used as a filter to decide if a candidate SNP is worth analysis in larger extend populations. The DNA MassArray throughput, measured in terms of genetic information output, depends on the chosen scale. Using microtiter plates and 8-channel pipettes the analysis of 192 genotypes (two 96 well MTPs) a day is routine work. With the use of automated liquid handling stations and 384 MTPs the throughput has been increased by a factor of about 30 (not including multiplexing capabilities). An automated process line (APL) has been developed to further increase the throughput up to an industrial scale. The APL integrates biochemical reactions including PCR set-up, MassEXTEND reaction and sample conditioning into a fully automated process.

5 Summary and Future Perspectives MALDI TOF mass spectrometry is a valuable tool for modern bioanalytical tasks in protein, carbohydrate, and nucleic acid analysis. MS-based techniques have evolved to become a routine high throughput device for analyzing genetic variations through the introduction of automated chip-mediated sample scanning. With a broadening of applications especially in the field of DNA analysis and a further automation in protein analysis MALDI MS is on track to become the gel electrophoresis of the 21st century.

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55. Lindblad-Toh K,Winchester E, Daly MJ,Wang DG, Hirschhorn J, Laviolette JP,Ardlie K, Reich DE, Robinson E, Sklar P, Shah N, Thomas D, Fan JB, Gingeras T, Warrington J, Patil N, Hudson TJ, Lander ES (2000) Nat Genet 24:381 56. Underhill PA, Jin L, Lin AA, Mehdi SQ, Jenkins T, Vollrath D, Da RW, Cavalli-Sforza LL, Oefner PJ (1997) Genome Res 7:996 57. Gonzalez E, Bamshad M, Sato N, Mummidi S, Dhanda R, Catano Cabrera S, McBride M, Cao XH, Merrill G, O´Connell P, Bowden DW, Freedman BI, Anderson SA, Walter EA, Evans JS, Stephan Clark RA, Tyagi S, Ahuja SS, Dolan MJ, Ahuja SK (1999) Proc Natl Acad Sci USA 96:12004 58. Bertina RM (1997) Clin Chem 43:1678 59. Larrey D (1989) Hepatology 10:168 60. Ross P, Hall L, Smirnov I, Haff L (1998) Nat. Biotechnol. 16:1347 61. Weaver T (2000) Trends Genet. In press 62. Braun A, Little D, Köster H (1997) Clin Chem 43:1151 63. Braun A, Little D, Reuter D, Müller-Mhysok B, Köster H (1997) Genomics 46:18

Received: June 2001

CHAPTER 1

Sequencing by Hybridization (SBH): Advantages, Achievements, and Opportunities Radoje Drmanac · Snezana Drmanac · Gloria Chui · Robert Diaz · Aaron Hou · Hui Jin · Paul Jin · Sunhee Kwon · Scott Lacy · Bill Moeur · Jay Shafto · Don Swanson · Tatjana Ukrainczyk · Chongjun Xu · Deane Little Callida Genomics, 670 Almanor Ave., Sunnyvale, CA 94085, USA. E-mail: [email protected]

Efficient DNA sequencing of the genomes of individual species and organisms is a critical task for the advancement of biological sciences, medicine and agriculture.Advances in modern sequencing methods are needed to meet the challenge of sequencing such megabase to gigabase quantities of DNA. Two possible strategies for DNA sequencing exist: direct methods, in which each base position in the DNA chain is determined individually (e.g., gel sequencing or pyrosequencing), and indirect methods, in which the DNA sequence is assembled based on experimental determination of oligonucleotide content of the DNA chain. One promising indirect method is sequencing by hybridization (SBH), in which sets of oligonucleotides are hybridized under conditions that allow detection of complementary sequences in the target nucleic acid. The unprecedented sequence search parallelism of the SBH method has allowed development of high-throughput, low-cost, miniaturized sequencing processes on arrays of DNA samples or probes. Newly developed SBH methods use DNA ligation to combine relatively small sets of short probes to score potentially tens of millions of longer oligonucleotide sequences in a target DNA. Such combinatorial approaches allow analysis of DNA samples of up to several kilobases (several times longer than allowed by current direct methods) for a variety of DNA sequence analysis applications, including de novo sequencing, resequencing, mutation/SNP discovery and genotyping, and expression monitoring. Future advances in biochemistry and implementation of detection methods that allow single-molecule sensitivity may provide the necessary miniaturization, specificity, and multiplexing efficiency to allow routine whole genome analysis in a single solution-based hybridization experiment. Keywords: Sequencing, Hybridization, Oligonucleotide, Arrays, Ligation

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Initial SBH Tests on Probe Arrays . . . . . . . . . . . . . . . . . . Use of Non-Universal Arrays of Probes . . . . . . . . . . . . . . . Hybridization Combined with Polymerase . . . . . . . . . . . . . Hybridization Combined with Ligase: Combinatorial Probe Scoring A Combinatorial SBH Blind Test . . . . . . . . . . . . . . . . . . . De Novo Sequencing, Comparative Sequencing and Genotyping Using Combinatorial Ligation and the HyChip System . . . . . . .

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1 Introduction 1.1 Direct and Indirect DNA Sequencing

Accurate DNA sequencing is a crucial procedure in modern biological, medical and agricultural research. Traditionally, DNA sequencing has been done by direct, base-by-base analysis, with each new base determination built on the results of many previous sequencing steps (Fig. 1). Such direct methods are effective but result in a gradual accumulation of errors in the sequencing process. Extreme experimental precision may help minimize these errors, but generally direct sequencing methods are restricted to relatively short (usually 300–1000 bases) DNA targets to maintain accuracy. This article discusses sequencing by hybridization (SBH), an indirect sequencing approach that avoids many of the shortcomings of the direct methods. Direct sequencing techniques involve a variety of synthesis, degradation, or separation techniques, and include the traditional Sanger [1], pyrosequencing [2, 3] and exonuclease methods [4], as well as direct visualization approaches [5–8]. In the Sanger method, DNA synthesis is randomly terminated at each base pair, creating a wide range of fragments that are then separated by gel according to length and scored. In the pyrosequencing method, polymerase-guided incorpo-

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a

b Fig. 1. DNA sequencing strategies. Sequencing methods are either direct or indirect, depend-

ing on the type of experimental data obtained from a DNA target. a Direct methods involve determination of which character or base is present in a particular position in the DNA string. There are two major types of direct sequencing. The first involves sequence-specific DNA degradation, synthesis, or separation followed by base-by-base assignment; the second involves direct chain visualization. b Indirect sequencing methods involve determination of which substrings of bases are present or absent in the DNA target. The first of two indirect approaches involves fragmentation of target DNA, followed by separation and identification of the resulting oligonucleotides. Sequencing by hybridization (SBH), the subject of this article, is the second indirect method. It involves sequence-specific pairing of DNA targets with complementary oligonucleotide probes of known or determinable sequence

ration of each base is detected by measuring the pyrophosphate released in consecutive cycles. In the exonuclease approach, a single molecule of target DNA synthesized with fluorescent-tagged bases is degraded by exonuclease. The consecutively released nucleotides are then scored with a very sensitive fluorescence detector.An interesting new class of direct sequencing methods relies on base visualization, using either scanning-tunneling microscopy [5–7] or nano-pores [8]. The main technical difficulties associated with direct base visualization are incorrect base differentiation and low speed. Indirect sequencing methods such as SBH have several unique advantages and have become important tools in deriving accurate DNA sequence information. Indirect sequencing methods are based on determining the oligonucleotide content of a target nucleic acid and do not require determination of base position information experimentally (Fig. 1). Oligonucleotide content may be obtained in two principal yet unrelated ways, by fragmenting the DNA and separating the resulting oligonucleotides based on physiochemical properties, or by specific hybridization of oligonucleotides to complementary sequences present in the test DNA. The fragmentation method was actually the first nucleic acid sequencing method [9–11], developed before the currently used Sanger gel-based method. Sequencing by hybridization was first proposed in 1987 [12, reviewed in 13]. In SBH methods, numerous oligonucleotides are tested for their ability to hybridize to a target DNA. Each oligonucleotide that successfully hybridizes to the target indicates the presence of a complementary DNA sequence within the target, revealing a small piece of information about its sequence. The complete sequence

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is then determined by compiling the results of many such hybridization tests. The SBH method has the potential to accurately and inexpensively sequence very long nucleic acids (>10,000 bases) in a single reaction. 1.2 History of Sequencing by Hybridization

Scientists from many disciplines helped construct the intellectual framework that led to the development of SBH in 1987 [12]. In 1960, Doty observed that, when heated in solution, double-stranded DNA “melts” to form single stranded chains, which then re-nature spontaneously when the solution is cooled [14]. This result suggested the possibility of using one piece of DNA to recognize another. In the late 1970s, researchers discovered ways to efficiently synthesize large quantities of short, sequence-specific DNA molecules by stepwise addition of nucleotides to columns [15]. At the same time, Bruce Wallace used oligonucleotides as “hybridization probes” to confirm the existence of complementary sequences in target DNAs, and demonstrated that they can accurately detect single base mutations in defined DNA targets dot-blotted on membranes [16]. In 1986, Lehrach proposed using a small set of oligonucleotide probes for cosmid clone fingerprinting [17]. At about the same time, PCR was developed by Mullis [18]. These early experiments provided basic research tools that were later fundamental in developing methods for fingerprinting and sequencing DNA by oligonucleotide hybridization. Sequencing by hybridization was first proposed and patented by Drmanac and Crkvenjakov in 1987 [12]. They proposed that the sequence of a DNA fragment could be determined by a hybridization experiment in which the target DNA was exposed to a set of oligonucleotides under conditions that favored full match hybridization. The sequences of the subset of positively hybridizing probes would then be used to determine the target sequence. One format proposed by the researchers was hybridization of labeled probes or various probe pools to large arrays of clones as a method for sequencing complex DNA samples. They presented an algorithm for reconstruction of DNA sequences as complex as the human genome [19].As with the PCR method, SBH dramatically expanded potential applications of the DNA hybridization reaction. Additional research studies followed in the SBH area. Bains and Smith demonstrated sequence reconstruction in a simulated assembly process using 256 specially designed 6-mers with two degenerate base positions in the middle (gapped probes) [20]. In a 1988 patent application, Southern proposed a method for combinatorial in situ synthesis of complex oligonucleotide arrays on glass [21]. He proposed that DNA samples could be hybridized to the probe array for mutation detection and complete sequencing. Lysov and colleagues also proposed DNA sequencing by hybridization to probe arrays, in this case using arrays of oligonucleotides deposited on a solid support [22]. Macevicz proposed using a small probe set having a binary probe design involving a specific base (i.e. adenine) and a degenerate “non-A base” (i.e., a mix of T, C, and G) [23].

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2 Principles of SBH 2.1 Proper Probe Length for SBH

When an oligomer of length n hybridizes specifically with a target DNA it reveals the existence of one or more complementary n-mer sequences within the chain. A particular n-mer sequence occurs roughly once in every 4n/2 base pairs of double-stranded DNA. This statistical ratio helps define appropriate design parameters for SBH experiments [19]. Long DNA molecules require longer probes, to avoid situations in which every probe has one or more complementary sequences in the target, leading to complete loss of specific information. Likewise, short probes (<5 bases) bind so frequently to most targets that they provide little or no useful information. Very long probes, on the other hand, occur so rarely that without prior sequence information to guide probe design their use is too inefficient for most standard SBH procedures. Very large sets of long probes may be used, but this approach becomes increasingly difficult as tens of thousands of individual probes must be synthesized and arrayed. Such large probe sets may require special methods of parallel synthesis and hybridization (see Sect. 5.1, “Hybridization Technologies”). In general, SBH experiments may use probe sets of length 5 to 25 bases, though some applications may use longer probes. SBH methods can accommodate nucleic acid targets of vastly different lengths, ranging from a few bases to hundreds of megabases, depending on the experimental design [13]. Probe pools ranging in size from a few to tens of thousands of probes can also be used to minimize the number of hybridization experiments required, a method that is especially effective for shorter target nucleic acids [12, 20, 23–26]. 2.2 Oligonucleotide Overlap Principle

In an SBH experiment involving a complete set of n-mer probes (i.e., all possible probes of length n), each DNA base is redundantly read by n overlapping probes. Figure 2 illustrates how probe overlap can be used to assemble the sequence of the target DNA in an SBH experiment using 8-mer probes. In this example, each positive probe overlaps seven bases of its nearest neighbor, six bases of its second neighbor, etc. This overlap principle allows determination of sequences that are much longer than the length of each probe by comparing and aligning n–1 or fewer overlapping bases that are shared by the probes. It is important to note that a full n–1 overlap between probes is not required to determine nucleic acid sequences; shorter overlaps may suffice, allowing for the use of incomplete probe sets. An additional advantage of probe overlap is that the effect of random errors is minimized because each base is “read” by multiple probes. Potential ambiguities in sequence resulting from the existence of long tandem repeats (e.g., (CA)30) and n–1 or longer repeat regions can be re-

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Fig. 2. Probe overlaps and assembly branches. A A sample sequence containing known primer sequences PPPPPPP at each end and two 7-mer repeat sequences is shown. B Assembly using overlapped probes is initiated at the 5′ end by first selecting a positive probe that corresponds to the primer sequence. Sequence extension continues in the 3′ direction until the first branching point, where two possible options for further extension occur (GGTCCCTc and GGTCCCTa). The branching process (C) leads to many alternative sequences. Requiring that the sequence must end with the 3′ primer and that sequence sub-fragments between branching points occur only once results in assembly of two possible sequences (D), which differ in placement of CAA and AAT sub-sequences

solved by additional experimental treatments such as the use of longer oligonucleotides, etc. The process of sequence assembly from the set of oligonucleotide sequences detected in the test sample is a unique feature of SBH and other indirect methods, and allows sequencing of DNA targets that have been randomly fragmented into pieces only slightly longer than probes. It also allows researchers to sequence mixtures of fragments from different DNA molecules, provided that the total length of all segments is not too large for the given set of probes. This characteristic is an important advantage, allowing diagnostic sequencing of diverse gene segments scattered across the genome that have been amplified in a single multiplex PCR reaction.

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2.3 Branching Points and Multiple Assembly Solutions

Determining a long DNA sequence from a large set of probes may pose unique technical challenges. One of these is the issue of “branching ambiguities”, which may occur when three or more probes that share a common end sequence hybridize to independent sites within the target DNA. For example the probes GATAC, ATACA and ATACG all share the common four-base sequence “ATAC”. These three probes represent a “branch point”, a region where sequence determination can proceed in two possible directions.Without additional sequence information it is impossible to determine whether the sequence at this branch point continues as GATACA or GATACG. Fortunately the only information missing in such a situation is the correct order of otherwise correctly assembled sequence sub-fragments between branching points. This information can be obtained using various experimental or computational DNA mapping techniques. Figure 2 shows a common example of a branching ambiguity that results in two equally plausible sequences [19]. The frequency of branching points depends on probe length, target length, and bias in base content of the target DNA. For random sequences, unique assembly is expected over approximately 90% of the time for DNA targets 200, 400, 800, 1,600 and 3,200 bp in length using probes 8, 9, 10, 11, and 12 bases long, respectively [19]. Longer probes appear proportionally less efficient than short probes, because each base added results in four times as many probes but only doubles the effective DNA sequence length. This paradox results from the test requirement that a single long sequence solution must be produced. Much longer DNA or RNA samples can be analyzed if instead the goal is only to assemble a target sequence in the form of a set of sub-fragments of a given average length. For example, a set of 12-mer probes may be able to sequence over 15 kb of DNA (instead of only 3.2 kb) if 30 sequence sub-fragments of length 500 bp (a typical gel read length) are assembled instead of a single continuous 15 kb DNA molecule. It is important to distinguish the actual number of probes in a complete probe set used in an SBH experiment from the actual number of independent probe hybridization measurements that are needed in the experiment. The number of hybridizations in an SBH experiment can be significantly reduced by using carefully designed probe pools. For example, a set of 16 million 12-mers is typically required for de novo sequencing of an unknown 3 kb piece of DNA. Fortunately a set of 16,384 oligomer pools (each containing over one thousand 12-mer probes) can be used effectively instead. Effective pool size can be increased until approximately one pool in five contains a positive probe for the DNA sample tested [25]. Long probe sets are not the only method of resolving branching ambiguities in long DNA targets. Many different experimental or data processing approaches have been suggested, including competitive hybridization [12], overlapping clones [12, 19], contiguous stacking hybridization [27], and simultaneous sequencing of similar genomes [28]. The use of closely related reference sequences (usually >90% identity) is also useful for eliminating branching point ambiguities. Such applications include sequencing of genes from related species, such as

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human and primate DNA or highly conserved ribosome genes from different bacterial species. Additional procedures in which reference DNA sequences are used to guide analysis of hybridization results include complete re-sequencing of targeted human genes from different individuals, complete re-sequencing of HIV and other pathogen genes and genomes, or SNP discovery experiments focused on detection of single-base changes. SNP discovery requires the greatest accuracy since rare polymorphisms or mutations (those occurring at less than 3% frequency) are usually present in the population as heterozygotes. In addition to using reference sequences to guide sequence assembly of the test sample, probe hybridization data obtained on one or more reference or control samples may be compared with test sample data to increase the confidence of the new base calls [12, 29–31]. 2.4 Universal and Target-Specific Sets of Probes

Universal sets of probes are designed using simple combinatorial and statistical principles, without guidance from prior knowledge of any specific gene or DNA sequence. Such probe sets may contain all possible probes of a given length, or a smaller subset of these, chosen without bias toward any specific gene sequence. An example of an incomplete universal probe set might be the set of all non-complementary probes (50% of all possible probes) used to analyze samples of denatured double stranded DNA. Another option might be a minimal set that covers each base in any sequence with at least one probe. This can be accomplished using a subset of about 3000 of the 16,000 possible 7-mer probes (unpublished data). Partial sequencing, in which only certain segments of the target DNA are determined by overlapping probes, requires even fewer probes. Universal probe sets may be used to sequence DNA from any organism, gene or mutant, and are suitable for many different applications, including de novo and comparative sequencing, genotyping, and diagnostics. The probe sets selected can be optimized to the particular research task. Complete probe sets provide the most accurate analysis, because every target sequence, known or unknown, is covered by a maximal number of overlapping sequences. Non-universal probe sets are chosen from larger universal sets on the basis of known sequence information from a DNA molecule of interest. In creating nonuniversal probe sets a variety of selection rules may be used. One common option is to use all (or a subset) of overlapping probes of a given length that are fully complementary to a known DNA sequence [31, 32]. Such sets can be further expanded by including probes that differ by one or a few bases from the known sequence [30, 32]. Because only a specific small fraction of the millions of possible probes is required, non-universal probes may be longer than those used in universal sets. This approach allows researchers to detect the presence of a known sequence in a sample, determine presence/absence and position of mutations, or discover novel SNPs or single base mutations. The main disadvantage of non-universal probe sets is the need to prepare a new set for every target.

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2.5 Probe Design and Hybridization Specificity

During the past three decades, numerous experiments have demonstrated the specificity of probe hybridization [33–35]. Reproducible hybridization data, with a high average ratio of full match to mismatch scores at every base position, facilitates sequence determination using SBH. Obtaining high discrimination for long probes is more difficult, especially for those with mismatched bases at the ends of the probe. Among all eight possible mismatches G/T and G/A are the most difficult to discriminate, especially when they occur in certain 3-mer sequences (CGC/GTG and GGC/CAG) [36, 37]. In most SBH experiments, probe sets are deliberately designed to improve discrimination. The use of probes shorter than 25 bases inherently enhances discrimination. In addition, SBH experiments may use probe molecules with an informative oligomer core of length n surrounded by a few degenerate (variable) bases, e.g. Nx– Bn –Ny , which helps enhance discrimination [12, 19, 31]. Using degenerate bases to surround a fixed oligomer core minimizes the problem of end mismatch discrimination because the fixed core is internalized within the probe. Degenerate probe ends provide an additional discrimination advantage. For a full match probe (i.e., all bases in the oligomer core match a sequence in the target DNA), most of the probe molecules in the pool will have one or a few mismatched bases among the degenerate base positions. For a single-mismatch probe, all probe molecules will have one additional mismatched base. Our research team and others [38] have shown that the discrimination observed between single-mismatch probes and those with two mismatches may be higher than that between full match and single-mismatch probes. In addition to changes in probe design, hybridization conditions can maximize full match/mismatch discrimination and enhance detection of signals from short probes that form weak hybrids. Based on thermodynamic and kinetic considerations, Drmanac et al. (1990) predicted that the most favorable conditions for successful discrimination in hybridization experiments are low wash temperature, long wash times, and an excess of initial hybrid [39]. The researchers demonstrated experimentally that these conditions allowed accurate discrimination between full match and single-base mismatch probe/DNA pairs, thus confirming that general hybridization conditions may be used in most SBH experiments. In 1991, Strezoska et al. used selected octamer probes under such hybridization conditions to accurately assemble a 100-base DNA sequence. The redundant hybridization data obtained from the set of overlapping octamers easily compensated for low error rates observed in the experiment [32]. Differences in base composition can affect hybridization efficiency in probes of identical length [40], as would be expected based on the observed differences between delta G values of various dinucleotide pairs [41, 42]. Such effects can impact hybridization strength as much as changing probe length by several bases. A variety of experimental strategies have been tested to minimize this impact of base composition on hybridization efficiency [12]. These include using tetramethyl ammonium chloride for probes longer than 14 bases [43], changing probe

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length by adding one to four degenerate [19, 29, 31] or universal [44–46] bases, using appropriate base analogs [47], or simply adjusting hybridization conditions for different probe types. Probe sequence, target/target hybridization [48, 49], stacking interactions [40], experimental imprecision and other factors influence absolute hybridization scores. Due to these factors the full match scores for some probes can be lower than the mismatch scores of others. Because of these uncertainties, control samples or groups of sequence-related probes [12, 29–32, 39, 50] are used to evaluate and transform absolute signals into relative values, which are then used in sequence determination. In SBH experiments, actual hybridization signal data may be first used to distinguish positive (full match) from negative probes (“probe call”). These assignments are then used in the sequence assembly step to perform a “base call.” However, an actual “probe call” step (assigning which probes are positive) is not necessary. A more general approach is to determine a score or probability that a given sequence is correct based on the scores of overlapped probes that fully match the suggested sequence vs. probes that have one or more mismatches for the same sequence. 2.6 Highly Parallel Data Collection in Different Formats

A hybridizing probe in an SBH experiment has completely random access to the data set (sequence), locating matching sequence strings anywhere within the target. This random access allows efficient parallel data processing, with the potential for thousands of probe-DNA hybridizations to be conducted simultaneously on a probe-or DNA-array or many other ways. One approach is to hybridize one labeled probe or probe sets at a time with arrays of samples [see, e.g., 12, 19]. Alternately, a single labeled sample can be hybridized with an array of probes [see, e.g., 12, 21, 22]. Furthermore, arrays can be designed in a predefined pattern of samples or probes or in a random collection of discrete particles that can be decoded at a later time [51]. Recent development of arrays on fiber optic bundles by Ilumina Inc. is very promising [55]. In addition to single component arrays, hybridization reactions can be performed with both components in solution in multi-well plates, especially if multiplex labeling of samples or probes is used [12]. Hybridization in solution requires efficient separation of hybrids from non-hybridized labeled components or a homogenous assay in which physically measurable changes occur upon hybridization, such as reduced fluorescence quenching when a probe hybridizes to DNA [53], or energy transfer between tags of two probes hybridized at a short distance [54]. The use of encoded micro or nano-tags and multi-well hybridization experiments will be discussed in a later section of this paper (see “Future Research Directions”).

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3 SBH Studies on Arrays of Samples 3.1 SBH on DNA Arrays

SBH methods are ideally suited to microarray technology due to their inherent potential for parallel sample processing. One promising SBH method involves exposing arrays of DNA samples to a single probe or probe pool [12, 19]. An important advantage of using a DNA array rather than a multiple probe array is that all resulting probe-DNA hybrids in any single probe hybridization are of identical sequence. This feature allows experimental conditions to be optimized for each hybrid type. In addition, using arrays of samples requires very simple sample preparation. Arrays of samples are routinely used at Hyseq, Inc. and in other laboratories [55–58] to screen thousands of cDNA samples for gene identification. Selected sets of about 300 7-mer probes are used for such screening. Over 55,000 individual samples and controls can be spotted to a single 16¥24 cm Genescreen membrane. Automated production lines are used to handle the large number of samples and hybridizations. For complete sequencing of 1–2 kb samples, approximately 10,000 probes or probe pools have to be tested, requiring 100 hybridization cycles on 100 replica arrays of DNA samples (see Sect. 3.3). 3.2 Blind De Novo Sequencing Test on Arrays of Samples

Drmanac et al. confirmed the accuracy of SBH in a blind test in which traditional gel methods and SBH were used to sequence three homologous primate T-cell DNA fragments [29]. The DNA targets, derived from human and rhesus monkey species, were 92–94% similar in sequence. The experiment used a pre-selected incomplete probe set that was carefully designed to test discrimination between full-and partial-match probes, and to distinguish very similar DNA targets from one another. In this study, one research team sequenced three 2-kb T-cell loci by the dideoxy gel-sequencing method. This information was then used to assemble a list of 272 octamer probes that would theoretically allow a second team to accurately sequence 116 bp regions of these clones by SBH. The selected probes contained about twice as many non-matching as matching probes and also contained 8-mers matching a fourth primate species not included in the test. The probes used in this experiment were actually pools of sixteen 10- and 11mer probes containing two degenerate (variable) bases surrounding a specific 8mer or 9-mer core. The longer probes increase hybrid stability, improving signal intensity and discrimination. Positive and negative DNA controls were also included, to allow calibration of the hybridization signals for the amount of DNA in each spot and to measure background signal. The DNA sequences of the three unknown targets were precisely determined after hybridization with only 156 of the 272 probes. Both SBH and traditional gel

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methods gave identical sequence results for all target DNAs in the study. The average discrimination value (ratio of signals obtained from full match and singlebase mismatch probes) was over ten in this study. False positives occurred infrequently and were generally due to probe hybridization with vector DNA or summed signals from two or more single-base mismatch probes. 3.3 Sample Arrays for Novel Polymorphism and Mutation Discovery

As genetic screening and gene therapy become increasingly important medical procedures, sequencing methods are needed that can rapidly and accurately detect mutations in patient DNA samples. Mutation detection by traditional gel sequencing methods is accurate but generally too slow and expensive for widespread use. In contrast, SBH using arrays of DNA samples provides an efficient, cost-effective way to screen large numbers of patients for individual genetic variations. Drmanac et al. [31] confirmed the accuracy and speed of SBH arrays in detecting mutations in a medically important gene. The researchers used in situ mutagenesis to create a variety of insertion, deletion, and substitution mutations in a single p53 clone. PCR-amplified DNA samples from these mutants were then spotted directly to membranes without further purification, a protocol that could be applied to any patient DNA sample. Replica arrays were created containing all p53 samples and a variety of positive and negative controls. In a blind experiment, 7-mer probe sets were then used to sequence all of the mutated p53 samples. The researchers used two probe sets in the p53 experiment, a complete set that matched all possible 7-mers and a smaller set that matched only the 7-mers present in the p53 reference sequence. Mutations were detected by comparing the average hybridization scores obtained for mutant and reference DNA samples using the reference set of probes. Figure 3 is a typical mutationscanning graph, a plot of the ratio of hybridization strength of sample (S) vs control (C) DNAs as a function of base position. When test and control DNAs are identical in a given region, S/C ratios are expected to be one. When a mutation occurs in a test DNA, the S/C ratio for the mutation scanning graph drops dramatically due to poor hybridization of the reference probes with the mutant DNA. This situation is seen at base positions 143 and 157 in Fig. 3. The exact sequence in the mutation region is then determined by hybridizing the complete probe set. A total of 13.2 kilobases of p53 DNA were sequenced with 100% accuracy in this experiment. The most difficult sequence assignments occur in regions where a single base is repeated several times in a row. When a point mutation occurs in one of these regions, only a few overlapped probes differ between the two sequence variants. Longer probes were used successfully in such cases. One advantage of the SBH process when large sets of probes are used for mutation detection is that multiple, independent reads of both DNA strands help ensure accurate sequence determination. In this study, five replicate samples of one p53 gene were amplified and sequenced independently to confirm the reproducibility of the SBH process. The coefficient of variation for median S/C values

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Fig. 3. A mutation-scanning graph showing two mutations in a p53 gene. Mutation scanning graphs are used to detect mutations in test samples relative to control (reference) DNAs. The S/C ratio represents the ratio of probe hybridization signals obtained for sample and control (reference) DNAs. For mutation scanning graphs the probe set used corresponds to the reference sequence. If the test sequence is identical to the reference, the median S/C ratio of seven overlapping probes will be close to 1, if a mutation is present in a given region, the median S/C ratio drops substantially because the reference matching probes do not bind to the mutated target DNA. In the mutation-scanning graph the flat line corresponds to a mutation-free control DNA; the sample line shows two mutations, a four-base deletion at position 143 and a G to A substitution at position 157

at mutant sites was below 3% for all cases, which is considered well within standards for most clinical diagnostic studies. An identical experimental setup with 384 replica arrays on one membrane has been used successfully to genotype ApoE variants in samples from approximately 400 patients with various cardiovascular indications [59]. An accuracy of over 99% was achieved.

4 SBH on Arrays of Oligonucleotides 4.1 Initial SBH Tests on Probe Arrays

SBH analysis of a single complex nucleic acid sample may require tens of thousands of individual probe-DNA hybridizations. Such massively parallel experiments are ideally suited to microarray applications in which oligonucleotide probes bound to a solid support are exposed to DNA samples alone or in combination with nucleotide bases or additional probes in solution (see below). Probe

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arrays may be prepared by spotting pre-made probes [22, 60] on a properly prepared substrate [61] using a robotic station equipped with needles, capillaries or various non-contact pipetting devices such as piezoelectric controlled nozzles. Probes can also be positioned by electric force on a chip containing an array of individually addressable electrodes (Nanogen). Alternately, arrays can be prepared by in situ synthesis, in which the required bases are coupled sequentially using a variety of methods for spot-specific delivery of reagents. These include physical masking [21], ink-jet or bubble-jet printing nozzles (originally proposed by Southern [21], and now applied by Agilent, Protogen, Rosetta Inpharmatics, and Canon), photolithographic [62] or micromirror [63] methods for directing light on photosensitive nucleotide protection groups, and micro-stamping procedures [64]. In principle, quality control of spotted arrays is higher than for in situ synthesis, due to the very high quality synthesized probes used in their manufacture. In one implementation of SBH, a labeled DNA sample is exposed to a probe array, which is then washed to remove target DNA that fails to hybridize or that forms unstable mismatch hybrids with bound probes. The labeled, hybridized samples that remain bound to each spot are then measured by a suitable detection device, such as a scanner. DNA sequences can be assembled from the hybridization results. The probe-DNA hybrids formed in such probe arrays have many different hybridizing sequences, which poses a technical challenge because pairs with different thermodynamic stabilities are formed and scored in a single hybridization reaction. Several sequencing demonstrations involving probe arrays have been performed, typically using small arrays of selected overlapping probes to sequence short DNA targets.An in situ prepared array of 256 8-mer probes containing only A and C bases was used to sequence a 20-base target [65]. Mirzabekov et al. also used small spotted arrays of overlapping probes to sequence synthetic oligonucleotide targets [66], and recently prepared complete arrays of all 6-mers [67]. Gunderson et al. [68] developed an advanced format 2 SBH procedure, previously proposed by Charles Cantor [69], in which complete arrays of 8-mer and 9-mer probes are attached to double-stranded anchors. Target DNA fragments with ends complementary to the probe are then ligated to the array. Target DNA of 1–2 thousand bases were re-sequenced with low error rates using this highly redundant array, which provides over 100 measurements per DNA base [68]. Longer samples (5–16 kb) were also analyzed. In addition to data obtained on test samples, similar hybridization data obtained for reference samples of known sequence were used in the base-calling process [68]. 4.2 Use of Non-Universal Arrays of Probes

Most current probe array SBH applications use non-universal sets of probes.Arrays prepared by in situ synthesis may contain 40 to 400 thousand probes. Such large arrays allow experimental designs featuring sets of probes suitable for large scale expression monitoring [70], SNP genotyping [71], or SNP/mutation discovery using a combination of full match, single-mismatch and single-base dele-

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tion probes matching each position in the reference sequence [72]. In SNP genotyping studies, over 1000 different SNPs may be scored simultaneously. The very large number of long probes allows SNP and single-base mutation discovery in very long DNA targets, demonstrated by an analysis of the entire 16.6-kb mitochondrial genome using an array of 135,000 15-mer probes [30]. In another application, an array of overlapped probes of varying lengths (up to 12 bases) was designed to match a series of tRNA sequences, to study how target secondary structures affect hybridization [49]. Further increases in the use and efficiency of non-universal probe arrays designed for specific sequences are expected. 4.3 Hybridization Combined with Polymerase

The use of DNA polymerase allows researchers to elongate already-hybridized probes, increasing the amount of sequence information obtained. Hybridized oligonucleotides may be extended by one or more bases using DNA polymerase and a set of four dNTPs tagged with different labels such as fluorescent tags [50, 69, 73, 74]. Deoxynucleotide bases must be sequentially blocked and unblocked in each elongation cycle, to prevent indiscriminate elongation of the probe. Each elongation cycle increases four-fold the number of oligonucleotide sequences that can be scored. Such sequence extension eliminates the need for initial probe labeling. Use of polymerase adds specificity at the extended 3′ end, but the process may increase mismatches at the 5′ end of probes. 4.4 Hybridization Combined with Ligase: Combinatorial Probe Scoring

Combinatorial ligation of two sets of short probes to create long probes is another powerful way to couple the hybridization reaction with an enzymatic or chemical covalent bonding step [50]. This combinatorial sequencing approach has several advantages over traditional SBH, particularly the ability to detect long complementary sequences in target DNAs without the use of large sets of long probes. For example, two sets of 4096 6-mers can be combined to score over 16,000,000 12-mers. Only a small number of manufactured probes are needed initially and each probe can be synthesized individually by standard chemical procedures to create large quantities of very high quality probes for use in millions of assays. By requiring simultaneous initial hybridization of two short probes instead of a single long one, the overall hybridization specificity is increased substantially. Covalent bonding provides a mechanism for accumulating sufficient signal to detect low-stability hybrids, which at any moment are present in very low quantities at the higher temperatures that are required to discriminate mismatches in very stable hybrids. Furthermore, such bonding helps minimize competition from target-target hybridization, a natural process that affects DNA interactions with probes. As covalent bonding of probes occurs, it removes target molecules from solution, driving target-target hybrids to dissolve to maintain equilibrium. Use of ligase also helps provide additional full match specificity, at least at the ligation site. In covalent bonding of adjacent probes, full match/mismatch dis-

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crimination is dependent in part on the equilibrium ratios of the corresponding hybrids during the hybridization reaction. Powerful low temperature discriminative washing procedures [39] cannot be used here to further increase specificity, but hot wash procedures after the ligation reaction can efficiently reduce background of non-specifically bound, labeled probes, thus increasing detection sensitivity. One very effective implementation of this combinatorial sequencing approach involves the use of two sets of short probes (usually 4 to 10 bases long) – one set bound to a support and another labeled set in solution [50, 75, 76]. Both sets of probes are hybridized to target DNA samples in the presence of DNA ligase. Bound and labeled probes that hybridize to the target DNA at precisely adjacent positions are ligated to form a labeled, support-bound long probe. Probes that do not hybridize to DNA samples at adjacent sites do not ligate and are removed in the wash process. The labeled, support-bound long probes formed by ligation are then scored by an appropriate detection device. The combinatorial SBH process has several unique advantages compared to simple probe array SBH experiments. Because a relatively small number of probes are used in each array, it is economical to create millions of identical arrays by spotting large quantities of high-quality probes prepared by standard synthesis. Furthermore, combinatorial SBH couples the capacity for spatial addressing inherent in probe arrays with the multiplex labeling advantages of probe pools in solution. This allows researchers to efficiently score millions of possible hybridization events for each sample using only a few thousand probes. For example, 10–100 subsets of differently labeled probes can be synthesized using a variety of fluorescent or mass tags. These subsets can then be combined to create pools that contain mixtures of 10–100 differently labeled probes, allowing independent scoring of 10–100 distinct hybridization events for each spot in the array. An additional advantage of using ligase-mediated covalent bonding of labeled probes to immobilized probes is the ability to use hot-wash procedures to conveniently and thoroughly eliminate background signal created by nonspecific adsorption of the labeled molecules to the support. In summary, combinatorial SBH methods may use sets of hundreds to millions of short probes to test millions to billions of 10- to 20-mer (or longer) oligonucleotides in a DNA sample. The method provides highly efficient, robust, and cost-effective methods of sequencing complex DNA or RNA targets. 4.5 A Combinatorial SBH Blind Test

Drmanac et al. confirmed the specificity and sensitivity of combinatorial SBH in a blind sequencing test using complex synthetic DNA targets [77, unpublished results]. Replica arrays of fifty 5–7-mer probes were spotted on nylon membranes and then hybridized in the presence of T4 ligase with a variety of specially designed DNA targets and 20–30 radioactively labeled 5–7-mer probes. The study, performed in collaboration with the National Institute of Standards and Technology (NIST), resulted in correct determination of 15 out of 16 homozygote and heterozygote sequences in complex DNA targets comprising several kilobases.

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Only a few overlapping probes were needed to sequence each site due to the high full match/mismatch discrimination observed in the experiment (an over 10-fold difference on average). 4.6 De Novo Sequencing, Comparative Sequencing and Genotyping Using Combinatorial Ligation and the HyChip System

Researchers at Hyseq, Inc. and Callida Genomics have developed the HyChip system, a universal combinatorial sequencing chip. The HyChip system consists of facing glass microscope slides containing four replica arrays of 1024 oligonucleotide probes consisting of all possible 5 base combinations. These bound 5-mer arrays are then exposed to DNA samples and a complete set of 1024 TAMRA-labeled 5-mers, combined in various pre-mixed probe pools containing 16 to 256 probes per pool (Fig. 4). With the combinatorial advantages of its ligation process, the HyChip universal system can score a complete set of over one million 10-mer probes per sample using only 2000 5-mer probes. DNA samples are generally prepared by PCR from genomic DNA or various clones. The complete probe sets used allow universal genetic analysis of any gene or DNA sample from any organism. In addition, the HyChip system allows efficient analysis of DNA samples of different length. Hyseq researchers have shown that a large number of probes with a single label type can be hybridized together as one pool without loss of information as long as relatively short DNA targets are used. When using complete sets of probes, about 4–8 hybridization scores are preferred per base of target DNA analyzed. To analyze 1000 bp of target DNA usually requires at least 4000 hybridization scores. This result can be obtained using 4 pools, each containing 256 labeled 5-mers. After adding target DNA to each pool the 4-pool set is tested on one HyChip having four replicas of 1024 immobilized probes, producing 4096 measurements. Probe pools work effectively, especially for short DNA targets, because only a fraction of fixed probes within the array give a positive result for each pool. In the case of a 1000 bp single stranded nucleic acid target sequenced using 256-probe pools, less than 1000 10-mers are full match, thus the remaining 3000, of 4000 pool scores, are negative. All major types of DNA analyses have been demonstrated using the HyChip system. De novo sequencing, the most challenging computational process, has been performed successfully using DNA targets of 100–1000 bases. The best results were observed with targets of about 500 bases in length. In a simulation experiment using 16,384 pools of 1024 12-mers, we demonstrated a 90% or greater success rate in assembling 3200 bp sequences. This result confirms that efficient de novo sequencing can be achieved with as few as five hybridization scores per base (16,384/3200) [25] using DNA targets that are over three times as long as those typically sequenced by traditional gel methods. Hyseq researchers have also performed comparative sequencing, re-sequencing and SNP discovery experiments on several human, bacterial, and viral (e.g., HIV) genes. Reference sequences, although not required, were used to aid in the assembly steps of these applications. It was, however, not necessary to obtain

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Fig. 4. Universal HyChip system: components and process. The system is composed of pentamer probe arrays on glass microscope slides and pools of TAMRA-labeled pentamers. Each slide has four replica arrays of 512 pentamers separated by a hydrophobic mask. Matching pairs of slides with different 512 probe arrays are sandwiched to form four capillary hybridization chambers, each chamber containing a complete set of 1024 pentamer probes.Arrays are prepared by spotting pre-synthesized probes (each coupled with a spacer and a primary amine group at its 5′ end) using an 8-channel spotting device. The probes are spotted as 200 um dots on glass slides that have been derivatized with 3-propylamino silane and with 1,4-phenylene diisothiocyanate. A complete set of 1024 TAMRA-labeled probes stored in vials is pipetted to create sets of 4, 8, 16, 32, or 64 pools containing 256 to 16 probes respectively. Each pentamer probe is present in only one pool in a given pool set. Target DNA, T4 ligase and buffer are added to each pool and the mixture is loaded by pipette onto the HyChip system, where it is drawn by capillary forces into a selected hybridization chamber. The hybridization/ligation reaction is performed for about one hour, allowing accumulation of positive 10-mer probe constructs. Each 10-mer formed consists of one labeled probe that has been ligated to an immobilized probe using the DNA target as a template. The presence/absence of each of the 1,048,576 possible 10-mers in the sample is thus tested in this combinatorial scheme. After incubation, the slides are hot-washed to remove unattached, labeled probes and DNA, and then read on commercial laser scanners (Axon, Luminex, Vertek). Hybridization results are then scored by image analysis software. A pattern of positive and negative control spots is visible in the figure

and use hybridization data from reference samples themselves, thus simplifying and reducing the cost of the sequencing process. Amplicons ranging in size from 100–10,000 bases in length were tested using sets of probe pools containing 16 to 256 labeled probes. By combining hybridization/ligation data for two complementary strands researchers achieved nearly 100% sequence accuracy in SNP discovery and complete re-sequencing studies of targets containing the sort of compound multiple mutations typically found in HIV samples.

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Sequencing complementary DNA strands independently minimizes the impact of pool-related false positive probes because the real positive probes for each complementary strand tend to fall, by chance, in different pools. Thus, the majority of real positive probes test positive in both experiments, while the majority of pool-related false positive probes test positive in only one experiment. We also found that sequence regions that produce low positive signals in one strand usually produce much stronger signals in the complementary strand, presumably because G/T-stable mismatches that cause target-target interactions in one strand correspond to very unstable C/A-mismatches in the complementary strand. In addition, having two data sets reduces experimental noise by providing twenty scores per base pair (10 per strand). An important advantage of redundant probe measurements per base is the ability to improve the accuracy of each base assignment or to declare some bases uncalled. With current non-optimized probe sets of uniform length and design (which produce hybrids of very different stability) single-strand sequencing generally result in about 2–3% uncalled bases. These are typically located in a few DNA regions, and they are almost always resolved when data from complementary strands or overlapping targets are included. The use of reference sequences to guide analysis of hybridization data allows researchers to process much longer DNA samples than can be handled in a de novo sequencing experiment. Using combinatorial SBH we have sequenced the entire single-stranded 5.4 kb genome of bacteriophage PhiX174, confirming the power of the SBH method to read much longer DNA molecules than can be processed by any other sequencing method today. In this experiment, 32 probe pools containing 1024 probes were used to generate about 33,000 individual measurements, or roughly 6 measurements per base. Furthermore, the HyChip system’s capacity to analyze complex DNA allows researchers to sequence mixtures of separate, unrelated DNA fragments, a unique advantage of the SBH process. This feature was confirmed by successfully sequencing a complex mixture of HIV, p53 and ApoB amplicons using the HyChip system. Hyseq researchers have also demonstrated efficient sequencing of several DNA targets that are AT rich (<40% G+C) or GC rich (>70% G+C). Some of these targets have proven very difficult to sequence by the Sanger dideoxy method due to stops or compressions in the sequencing process. Accurate identification of heterozygotes is also routinely achieved with data obtained from the HyChip system, as shown in Fig. 5. Interestingly, both correct bases at the heterozygous site scored as high as did single bases in homozygous sites. The high signals observed for both alleles allow separation from background noise and correct identification of the heterozygous bases. Another application of the universal HyChip system is genotyping, which involves detection of known allele sequences in a DNA sample, and is usually done by use of locus-specific primers and probes. In SBH genotyping studies, short DNA samples containing a polymorphic site are prepared individually or simultaneously in a multiplex PCR reaction.A single HyChip system with four labeled probe pools is sufficient to genotype 10–20 sample amplicons roughly 50 base pairs in length, providing the preferred 4 to 8 scores per base for the combined 500–1000 bases. Using the HyChip system, each amplicon is completely rese-

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Fig. 5. Sequencing a heterozygote site in the ApoB gene using the HyChip system. The base-calling graph of a region from 450–480 base position of a 700 bp ApoB amplicon is depicted. The base scores represent the average relative fluorescent signal of ten overlapping probes corresponding to each of four possible bases at each position. Capital letters represent reference bases of the ApoB gene. At every base position except 471, the three non-reference bases have a much lower score than that of the reference base. At base 471 both A and the expected reference G show almost identical high scores, with the other two bases showing low scores typical of an incorrect base

quenced, with each base in the polymorphic site (homozygote or heterozygote) read by 10 overlapping probes. Ligation of two complete sets of short probes offers advantages of universality, redundant testing of each allele with multiple overlapping probes, and eliminates the need for new probe design and test optimization with each new SNP. An additional option for genotyping is to use universal arrays and to select from a universal set of labeled probes a few probes corresponding to each polymorphic site. In this case a single pool of 128 labeled probes can test 32 SNPs, with four overlapping labeled probes corresponding to each locus. One HyChip system can be used to test a set of 32 SNPs in four individuals, or, using four specific probe pools, to test a set of 128 SNPs in a single sample. In summary, combinatorial SBH based on the universal 5-mer HyChip system has great potential for sequencing, genotyping, and polymorphism and mutation discovery.A universal 5-mer HyChip system could be used to monitor expression of a selected set of dozens of genes, or, if enlarged to 6–8-mer capacity, to sequence over 100 kb in one reaction, and to monitor gene expression. The HyChip combinatorial system allows testing of complete sets of millions of possible oligonucleotides in each sample, resulting in long, accurate DNA reads, and providing the universality and flexibility to analyze any sample, any gene, or any sequence variant.

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5 Future Research Directions 5.1 Hybridization Technologies

In SBH experiments, analysis of increasingly complex DNA molecules generally requires use of longer probes and larger probe sets. Practical analysis of long targets with complete probe sets requires that the probes chosen be long enough and pools small enough that less than half of the probes or pools tested result in positive hybridization. A DNA molecule of length L bases has approximately L positive probes. This imposes a minimum of 2L measurements (two per base); at least 50% of which should be negative. Note that the number of overlapping probes that read each base is equal to the number of bases in the probe, independent of target length and number of probes per pool. In general, hybridization data from four to ten probes (or probe pools) per base will yield proper assembly and high accuracy in base calling. By comparison, single-pass gel sequencing also requires four or more “scores” per base. A variety of different techniques can be used to further increase the throughput of SBH. Use of multiple probe or DNA labels may greatly simplify SBH experiments. For example, sequencing 1 Mb of DNA in a single reaction may use up to 10 million hybridization measurements. These may be obtained with a 10 million-element array, or with a 1 million-element array if ten different fluorescent labels are used in a combinatorial scoring approach (see Sect. 4.4). The required number of DNA or probe spots may be further reduced if a highly multiplex system of 100 or more tags is used, for example, by use of mass tags that can be identified by mass spectroscopy [79]. Currently, probe arrays or random arrays of micron size beads on optical fibers (Ilumina Inc.) may have effective feature spans of approximately 15 microns, permitting roughly 500,000 features per square centimeter. At such a density, sequencing an entire bacterial genome at once using a single label would require a large 10¥10 cm chip. Further reduction of feature size below 5 um would greatly enhance the potential of SBH arrays. Non-array methods may also be used to efficiently score large numbers of probes. Color-coded or otherwise distinguishable micro-beads may be used [51]. For example, Luminex has developed a flow-sorting detection system with 100 distinct beads encoded by specific ratios of two fluorescent dyes. In this system, a single small well containing 100 beads would replace 100 DNA or probe spots, a 1536 well plate would replace an entire array of 153,600 single color spots, and 60 plates would permit the 10 million hybridization measurements needed for analysis of 1 Mb of DNA. The development of multiplex systems containing 1000 or more encoded beads would obviously be of great benefit [51]. It may be possible to produce several thousand unique Q-beads (QuantumDot, Inc.), and virtually unlimited numbers of micro transponders with specific emission frequencies can be designed (PharmaSeq Inc.). Unfortunately, current micro-transponders are still quite large (250 um), and may ultimately prove to be too expensive for most research purposes. Another new SBH platform combines

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micro-fluidics with probe arrays (FeBiT, Inc., Motorola, Inc.) or bead assemblies [80] located in micro-capillary channels. The oligonucleotide probe scoring process may ultimately be miniaturized to involve detection of a single positive probe molecule in solution. Such a process would provide virtually unlimited multiplexing capabilities [81].A variant of this probe labeling and detection process is schematically presented in Fig. 6. Nano-barcode tags are prepared as chains of nano beads, other particles, or large molecules.An example with four different monomer units (combination of two lengths and two diameters) is depicted in Fig. 6. In this case one of four monomers is assigned to each base. To encode all N-mer probes, the barcode chain has to be N elements in length. Sets of one million 10-mers to one trillion 20-mers can be encoded and potentially analyzed in a single reaction. After hybridization, probes bound to the target may be separated from unbound probes and their barcodes read by the detector device. The advantage of three-dimensional nano-tags is the ability to identify single molecules when a tagged molecule passes between a pair of nano-electrodes. The flow of electrons between electrodes will be influenced differentially by the size and shape of different monomer tags, providing a specific signature. Large sets of tagged probes can be prepared in a sequential mix-and-divide synthesis scheme. An equimolar mix of four tagged monomers is split into four separate synthesis reactions to add A, T, C, and G to the oligonucleotide end and a corresponding base-specific tag on the tag end, resulting in 16 dinucleotides, each labeled with a specific dimer tag. By repeating n cycles of mixing and di-

Fig. 6. SBH in solution using three-dimensional nano-barcode tags. a Probe ATCG tagged with

a four-monomer tag passes between a pair of closely spaced 1–10 nm electrodes. b Readout of signal variation over time. The signal graph reflects length and diameter of probe molecule as a whole, without ability to distinguish individual bases, which are only about 0.3 nm in diameter.Alternately, the individual barcode tag units cause significant differences in strength or duration of the electronic signal. By using sets of four monomer tags, all possible n-mer probes can be identified by n-unit-long barcodes. Tens of millions of tagged probes can be prepared with only a few dozen coupling reactions in a mix-and-divide synthesis scheme

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viding while adding new base/tag pairs, all possible n-mer probes can be prepared in a series of 4n syntheses. Construction of all possible 20-mers would thus require 80 reactions. As with other SBH procedures, nano-tagged probes can be hybridized directly or extended by use of DNA polymerase. In addition, two mixtures of shorter probes (tagged at opposite 5′ and 3′ ends) can be ligated and efficiently analyzed by reading the two tags at the ends of the ligated oligonucleotides. Other researchers have also proposed the possibility of distinguishing natural bases directly using a similar principle, by passing DNA through membranes with nano-pores [8]. The main difficulties facing this process are the very small sizes of the nucleotide bases and the slight variations in size that exist between A and G purines and T and C pyrimidines. Nanotags allow engineering of monomers from materials that are several times larger than natural bases, with large differences in monomer size to assure quick and accurate detection. Development of SBH technology with single molecule sensitivity can lead to whole human genome analysis on a single tiny device, without need for DNA amplification or fractionation. The ability to rapidly read billions of tags or bases in parallel analyses using thousands of reading channels would facilitate large genome sequencing in a single day. 5.2 Hybridization Chemistry

The hybridization success of full match probes depends primarily on the differential stability of probe/target and target/target hybrids. Probes which form less stable hybrid duplexes not only produce inherently weaker signals, but are also less likely to compete with imperfect but longer target/target hybrids. Probe designs that increase hybrid stability can dramatically improve hybridization efficiency. For example, lengthening a probe by one base may produce a 10–20-fold stronger signal than can be achieved with a 10-fold higher concentration of the shorter probe (unpublished data). The challenge is to obtain optimal stability for all probes under the same conditions, because low efficiency probes do not produce strong enough full match signals, while probes with very stable full match hybrids also produce strong signals with mismatch targets. The ideal design solution would be a set of probes with the following characteristics: 1. equal hybridization stability among all possible full match probe-DNA pairs, 2. probe-DNA hybridizations that are stronger than competing DNA-DNA or probe-probe hybridization (even for DNA-DNA hybrids longer than probes), and 3. high full match/mismatch discrimination for all types of mismatches in all sequence contexts. Both peptide nucleic acids (PNA) [82, 83] and locked nucleic acids (LNA) [84] may increase hybrid stability, and have great potential to meet these conditions, unless non-standard binding produces an intolerably high level of artifacts. Unfortunately, the modified chemical backbone of such probes may limit use of en-

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zymes (such as polymerase, ligase or mismatch recognition enzymes) used for enhanced hybridization scoring. There are several ways to equalize the hybrid stability of DNA (or PNA and LNA) probes of differing base composition. These methods include the use of longer informative sequences to increase the binding strength of (A+T) rich probes, or the use of variable numbers of degenerate bases at the probe ends [19, 31]. Development of universal bases that form pairs with delta G values equal to those of regular A/T pairs would be desirable. Current universal bases [44, 45] usually pair with natural bases with binding efficiencies comparable to mismatch base pairs. One intriguing possibility would be to combine an LNA-modified sugar with an existing universal base to enhance the binding efficiency. In addition, nucleotide base analogs may be used, particularly to increase the stability of A/T pairs. Promising new options are: 1. The use of LNA-modified T or A bases, coupled with use of standard C/G bases, (which naturally bind more strongly than A and T), and 2. coupling special minor groove binders to A+T rich probes to stabilize their hybridization to complementary DNA [85]. On average, probes with roughly equal hybrid stability give similar discrimination values. To further minimize mismatch signals, especially of G/T pairs in certain surrounding sequences [36, 37], potential use of electronic stringency, [86] or mismatch recognition and/or cutting enzymes [87, 88] or chemicals [89] may be beneficial. In the case of combinatorial probe scoring (e.g. HyChip), use of selected [90] or specially designed ligase enzymes that are highly sensitive to mismatches within several bases of the ligation site may provide a solution that does not require any additional treatment. 5.3 Advanced SBH Applications

There are several important research and medical applications that demand extremely efficient and accurate DNA sequencing. These include: – Routine research sequencing of thousands of new bacterial and eukaryotic genomes, – Cataloging of rare genetic polymorphisms, by sequencing thousands of individual human genomes, – Complete resequencing of viral and bacterial genomes to identify drug resistance or virulence genes for infectious disease management, – Genetic screening and diagnostics, via complete sequencing of hundreds or thousands of the most medically important genes in infants, cancer or heart disease patients, etc., and – Complete genome analysis for medical prognostics and diagnostics, by genotyping millions of SNPs or completely re-sequencing the genomes of all newborn infants. Achieving these research and medical needs may require development of sequencing methods that can acquire over 1 GB of highly accurate, inexpensive

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sequence data per instrument per day. This throughput capacity, which is 1000–10,000-fold higher than currently available, will most likely require the use of integrated micro-fluidics to process genomic DNA samples without handling millions of amplified fragments in multi-well plates. The inherent parallelism and long read-length of SBH technology may be the only practical solution to these challenges.

6 Conclusion We live in a world of self-encoded, evolving biological systems, each of which possesses precise instructions for development and function recorded in its genomic DNA or RNA. Advances in the medical, biological and agricultural sciences are thus critically dependent on obtaining a large amount of accurate DNA and RNA sequence information quickly at the lowest cost. Despite great progress, current sequencing methods still limit the acquisition of and realization of the value of these large sequence data sets in modern research. Even though the entire human genome has been successfully sequenced there remains a great need for advanced new sequencing technologies. Efficient methods such as SBH have the potential to obtain accurate DNA sequence information at 1000-fold faster rates than are achieved today. This ultra-fast DNA sequencing would propel progress in the life sciences, with tremendous potential to improve overall human health and wellbeing. Acknowledgement. We thank Kathy Ashue, Rodney Bellot, Haibin Chen, Linsu Chen, Linh

Duong, Jenny Le, Charlene Lee, Halina Loi, Xinyu Mu, Kiran Mukhyala, Thutrang Nguyen, Helena Perazich, Khao Pham, Tami Pham, Tran Van, Christine Wong, Wei Wu, Linda Wu, Sean Sleight, Pouneh Tavakoli Han Wang and Sherry Zhang for their assistance throughout this project and Elizabeth Garnett for her organizational efforts and preparation of figures.

7 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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CHAPTER 6

Protein Array Technology: The Tool to Bridge Genomics and Proteomics Holger Eickhoff 1 · Zoltán Konthur 2 · Angelika Lueking 2 · Hans Lehrach2 · Gerald Walter 2 · Eckhard Nordhoff 1 · Lajos Nyarsik 2 · Konrad Büssow 2 1 2

Scienion AG, Volmerstr. 7b, 12489 Berlin, Germany. E-mail: [email protected] Max Planck Institute of Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany

The generation of protein chips requires much more efforts than DNA microchips.While DNA is DNA and a variety of different DNA molecules behave stable in a hybridisation experiment, proteins are much more difficult to produce and to handle. Outside of a narrow range of environmental conditions, proteins will denature, lose their three-dimensional structure and a lot of their specificity and activity. The chapter describes the pitfalls and challenges in Protein Microarray technology to produce native and functional proteins and store them in a native and special environment for every single spot on an array, making applications like antibody profiling and serum screening possible not only on denatured arrays but also on native protein arrays. Keywords: Protein Array, Expression Profiling, Automation, Serum Screening, Antibody Profiling, Protein Purification

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1 Introduction Each cell of a living organism contains the whole genetic information in the form of DNA molecules. The available golden path sequence of the human genome is known to be 3¥109 nucleotide base pairs in size, coding for a currently unknown number of genes. Although the DNA information is, with rare exceptions, identical in each cell, several hundreds of different known cell types do exist. The simplified view of a cell brings up specific populations of DNA and mRNA molecules that are translated into cell-specific populations of proteins.Although this is only a simplified representation, we are currently able to understand only a minority of the complex interactions in the cell machinery comprising DNA, RNA and proteins as major compounds. Genomic databases have enabled us to access, retrieve and process biological information. The determination of the genomic sequences of higher organisms, including humans, is now attainable, but represents only one level of genetic complexity. The determination of the expression profiles and protein profiles of certain cell types represents the next level of genomic complexity, equally important to sequencing. The interest of our research is focussed in finding all genes, their in vivo functions and the features of the corresponding proteins. Information about a gene’s expression is important for its potential exploitation.A gene’s expression and the corresponding protein level can be highly specific to a tissue, organ, cell type or disease and, as such, may be attractive as targets for the development of highly specific therapeutics and diagnostics. Even a gene of unknown function may have medical utility if its expression pattern can be determined. To achieve this goal, methods and technologies operating reliably with many samples in high-throughput and in parallel are major requirements. The human genome is sequenced, but only a minority of genes has been assigned a function. Automated technology allows for high-throughput, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridisation on arrays of oligonucleotides or cDNA clones. Having started from high-density filter membranes, cDNA microarrays nowadays are mainly used in chip or microscope slide format. In the past our group has shown that the same cDNA libraries used in gene expression analysis can be used for highthroughput protein expression and antibody screening on high-density filters and microarrays. Most importantly, these libraries connect recombinant proteins to clones identified by DNA hybridisation or sequencing, hence creating a direct link between gene catalogues and functional catalogues. Microarrays can now be used to go from an individual clone to a specific gene and its protein product or vice versa. Clone libraries have become amenable to database integration including all steps from DNA sequencing to functional assays of proteins. The medical application of this information is expected to lead to new generations of products in the diagnostics and therapeutics market. However, genes will only be useful for drug development and medical diagnostics if their functions are known. To tackle the current limitations in the medical use of

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genome information,“functional genomics” or “functional proteomics” are now under development as a new research and development area. With the introduction of automated technologies in the field of molecular biology and, especially, microarray technology, genome and gene expression analysis have been accelerated enormously. Microarray technology was enabled by the development of devices that can array biological samples at high density and with high precision [1]. Oligonucleotide and cDNA microarrays have become hot commodities, representing thousands of individual genes arrayed on filter or glass slide supports (The chipping forecast, Nature Genetics supplement 1999 [2]). To examine variation in gene expression, sets of oligonucleotides or complex probes, generated by reverse transcription of RNA from different tissues and cell lines, are hybridised on the arrays [3]. cDNA microarrays have already been used to profile human tissues like bone marrow, brain, prostate and heart [4] and complex diseases such as rheumatoid arthritis [5] and cancer [6, 7]. However, the DNA chip technology is still hampered by the lack of common quality standards that enable the comparison of results obtained in different laboratories and with arrays of different origin [8, 9]. Nonetheless, protein chips are already emerging to follow DNA chips as tools for automated and miniaturised functional analysis [10, 11]. Analogous to DNA microarrays, protein arrays offer the opportunity to screen thousands of immobilised biomolecules at a time, using steadily reduced amounts of sample.

2 From 2D Electrophoresis and Microtitre Plates to Microarrays of Biomolecules Two-dimensional gel electrophoresis separates proteins according to size and charge, therefore allowing the study of cell, tissue and even whole organism proteomes [12]. Until recently, however, the identification of the thousands of separated proteins used to be a major challenge. With the introduction of new and automated mass spectrometric protein identification procedures, the high throughput identification of the separated proteins is much simplified [13] and allows us to generate catalogues of expressed proteins in a cell or tissue of interest. Nevertheless, as the separated proteins are obtained in denatured form and in limited amounts, the expression of a protein of interest in recombinant form is usually required for functional characterisation. The classical array format in proteomics, the microtitre plate, is a well established and still widely used standard in medical diagnostics. To increase the number of samples and decrease reagent volume, the 96-well microtitre plate has been developed further to plates with 384 and 1536 wells, maintaining the original plate footprint. As it happened in DNA analysis earlier, the microtitre plate is now gradually being replaced by microarrays on flat surfaces such as glass slides (“chips”) or membranes. The format and the preparation of protein microarrays depends on the nature of the immobilised biomolecule and its application. While peptide arrays are manufactured synthetically directly on the support [14], proteins are delivered using either pin-based spotting or liquid microdispensing. To date, the most com-

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monly arrayed proteins are antibodies, since they are robust molecules which can be easily handled and immobilised by standard procedures without loss of activity.

3 Requirements for Protein Arrays For protein arrays, resources of large numbers of proteins, preferably in purified form, represent a major technical challenge. While in prokaryotes genomic fragments can be directly cloned into expression vectors, intronic sequences prohibit this strategy in eukaryotes. In systems such as human tissues, full-length cDNA clones have to be isolated before protein expression can be started. Highthroughput subcloning of open reading frames has been described [15] but remains a major difficulty if complete proteomes of higher organisms represent the region of interest. To overcome these problems, arrayed cDNA expression libraries, cloned in bacterial and yeast expression vectors, have been developed in our laboratory. These libraries are generated by standard DNA cloning procedures and characterised by oligonucleotide fingerprinting to be screened for the properties of their expression products. Furthermore, these libraries do represent an immortal source for large numbers of recombinant proteins [16–18]. In addition, expression libraries eliminate the need to construct individual expression systems for every protein of interest and, by arraying, the expression products of complete libraries can be characterised in parallel. On the other hand, a large proportion of clones do not express their insert in a suitable form, mainly due to cDNA fragments being fused to the vector-encoded start codon in the wrong reading frame. Therefore, non-expression clones have to be identified and removed from the library. To identify expression clones, hundreds of thousands of clones are arrayed on filter membranes and protein expression is induced. By detection of a His6-tag peptide fused to the protein products, desired expression clones are identified and re-arrayed into a non redundant daughter expression library. The protein filter array technology was further developed to increase spot density and to facilitate the arraying of purified proteins. Lueking et al. have used automated arraying from liquid expression cultures using a pin-based, flat-bed gridding robot [19]. For this purpose, 96 proteins of the human foetal brain cDNA library hEx1 [17] were expressed in liquid bacterial cultures, and solutions were spotted onto polyvinylidene difluoride (PVDF) filters, either as crude lysates or after purification by Ni-NTA immobilised metal affinity chromatography (IMAC). In all 4,800 samples were placed onto polyacrylamide-coated microscopic slides and simultaneously screened, using a hybridisation automat, applying minimal amounts of reagents (less than 100 mL antibody solution; A. Lueking, personal communication). Sharp and well-localised signals allowed the detection of 250 attomol or 10 pg of a spotted test protein (GAPDH, glyceraldehyde-3-phosphate dehydrogenase, Swiss-Prot P04406).

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4 Planar Immobilisation of Proteins The Lehrach laboratory mainly uses solid pins routinely for spotting nanolitre volumes of proteins although proteins can be delivered onto solid supports by either split pin-based spotting or microdispensing devices. In contrast to other techniques, solid pins are less sensitive to variation of sample viscosity than slit pins or microdispensing systems and are much easier to clean to prevent any cross contaminations [19, 20]. A ring and pin arraying device was used by MacBeath and Schreiber to produce a microarray of 10,800 spots of two distinct proteins (protein G and an FKBP12 binding domain), which were then specifically detected with fluorescently labelled IgG and FKBP12, respectively.Although the detection scheme is this article is really clever, it remains unclear why the authors did array 10800 replicates made of only two proteins [21].As an alternative to modified glass surfaces gel immobilisation matrices show high binding capacities and provide the proteins with a nearly (97% water in the buffer system) native environment. This is a key feature in protein array research and is important for the reactivity and specificity of the arrayed proteins. The technique of immobilisation is substantial both for effective concentration and orientation of immobilised proteins or antibodies on the surface. A variety of methods have been reported, including the adsorption to charged or hydrophobic surfaces, covalent cross-linking or specific binding via tags (e.g., His6, biotin/avidin system). The density of protein molecules immobilised on the support is mainly determined by the surface structure. A flat, two-dimensional surface offers less binding capacity than the three-dimensional structure of a filter membrane or a polyacrylamide gel layer. Mirzabekov and co-workers produced three-dimensional polyacrylamide gel pad microarrays providing a more than 100 times greater immobilisation capacity than two-dimensional glass supports, thus increasing the sensitivity of measurements considerably [22]. The gel pads are separated by a hydrophobic glass surface and provide a native, aqueous environment and can accommodate proteins of up to 400,000 Dalton in size [23]. Enzymatic activity of several enzymes like horseradish peroxidase, alkaline phosphatase and β-D-glucuronidase has been detected in these hydrogel pads. Prestructured surfaces consisting of hydrophilic spots on hydrophobic surfaces have also been reported for protein arraying [24, 25]. The hydrophobic surface prevents the aqueous drops applied to the hydrophilic spots from mixing and creates wall-minimised reaction vessels, where the interactions can be monitored in solution. In combination with state of the art microfabrication procedures, prestructured surfaces allow the introduction of three-dimensional microstructures on a chip, offering a number of additional options for experimentation like on-line monitoring of the interaction kinetics. Such microfluidic devices can be equipped with channels for transporting reagents to immobilised target molecules.At the present time microfluidic chips do certainly offer specific advantages over planar microarrays [26] but due to their complex production procedures and the high surface to volume ratio that represents a potential non-specific binding site for the analyte, their development and applications are still at an early stage.

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5 Detection of Molecular Interactions on Microarrays On DNA microarrays, hybridisation events are detected using fluorescently or radioactively labelled probe molecules [27]. A corresponding approach for the detection of protein-protein, protein-DNA and protein-small molecule interactions has been reported, the “universal protein array system” (UPA), consisting of filter membrane arrays of purified proteins [28]. Specific binding properties of the immobilised proteins on the low-density UPA arrays were demonstrated with various radiolabelled protein, DNA, RNA and small molecule ligands. By washing the membrane with different salt conditions, high-affinity protein-protein interactions could be distinguished. In addition to fluorescent dyes and radioisotopes, a wide range of detection options exists for protein and antibody arrays (reviewed in [29]). Unlabelled ligands can be identified indirectly by using a secondary antibody (sandwich assay). Alternatively to these non-competitive formats, various competitive assays, relying on competition of the ligand with labelled tracers, are in use. Protein chips for direct measurement of protein mass by matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) mass spectrometry have been described [20, 24, 30].

6 Applications of Protein Arrays A large variety of assays has been adapted to utilise protein microarrays. At its current state, the detection of immobilised antigens with antibodies is still the most common application. Protein and antibody arrays have been used for the selection and characterisation of novel antibodies from phage display libraries and for the identification of antigens (e.g., involved in autoimmune diseases). Phage display antibody libraries have been developed for the in vitro selection of antibodies as an alternative to animal immunisation (reviewed in [32], [53]). For this purpose, recombinant immunoglobulin gene libraries are cloned in phagemid vectors and antibody fragments are displayed as fusion proteins on the surface of bacteriophage (reviewed in [34]). Recently, protein arrays of the cDNA expression library hEx1 [17] were used to identify antigens recognised by randomly selected antibody fragments from a phage display antibody library [35]. Screening 12 different antibody fragments on an array of 27,000 expression clones, delivered four novel and highly specific antigen-antibody pairs. In a related approach, antibody arrays were used for the identification of specific antibody-producing bacteria [36]. For this purpose, bacteria containing phagemid selected from a phage antibody library by in vitro panning on chosen antigens were arrayed on filter membranes.After cell growth, antibody production was induced and specifically binding antibodies were captured and identified on a second, antigen-coated membrane. By screening 18,342 antibody clones at a time, highly specific antibodies were selected after just one round of panning. In autoimmune diseases self-reacting antibodies, i.e., produced against the organism’s own proteins and epitopes, play an important role in the clinical manifestation of the diseases. Therefore, the generation of an antibody profile of pa-

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tients with autoimmune disease is believed to be medically relevant and informative. Characterisation of autoimmune patient sera on protein chips would allow the diagnosis of autoimmune diseases based upon the presence of specific auto-antibodies. So far, for the identification of antigens recognised by auto-antibodies, sera were hybridised to uncharacterised gt11 cDNA phage libraries or to tissue extracts separated by 1D or 2D gel electrophoresis [37, 38]. The following characterisation of the identified antigens is labour intensive, also requiring expensive sequencing of the found proteins. Such characterisation resulted in novel functions attributed to these proteins, which can then be used as potential therapeutical targets [39]. To simplify the characterisation of auto-antibodies, serum can be applied to protein arrays containing large numbers of recombinant proteins of known identity. Moreover, using protein arrays will overcome the problems associated with protein level variation in natural tissue extracts and hence increase reproducibility. The application of protein chips allows us to determine the binding profile of the autoimmune antibodies of each patient and for each disease. Once disease-specific antigens are known, it is possible to create a diagnostic protein array. As shown by Lueking et al., apparently specific monoclonal antibodies (a-HSP90, a-b-tubulin) showed considerable cross-reactivity with other proteins following incubation on protein microarrays, consisting of 96 in liquid bacterial cultures expressed proteins of the hEx1 library [19]. In a way, this is not surprising, as antibodies are not usually tested against whole libraries of proteins. However, in immunohistochemical or physiological studies against whole cells or tissue extracts, this cross-reactivity of antibodies can lead to false interpretations. Therefore, the characterisation of the binding specificity of antibodies used extensively in diagnostic tools is of prime importance.

7 Outlook To achieve standardised microarrays carrying thousands of verified recombinant proteins, high-throughput methods for protein expression and purification are required. This has to be accompanied with a pipeline for the identification and verification of expression products. Initial experiments have shown that especially MALDI-MS is a powerful tool to monitor the quality of recombinant proteins. Combining protein expression and purification in array (microtitre plate) format with high-throughput protein mass determination by mass spectrometry leads to a large number of identified library clones and their corresponding expression products [17, 20]. This approach can be used either to identify unknown clones from expression libraries or to verify expression products generated at high-throughput. In addition mass spectrometric data allows us to compare recorded spectra from recombinant and native proteins, which results in unambiguous protein identification in, e.g., 2D protein gels. A very crucial part in protein array technology is played by the deposition of the proteins on a suitable surface. As described earlier, the nature of the surface with influencing parameters like charge, viscosity, pore size, pH, binding capacity, unspecific protein binding, etc. is essential for the generation of protein arrays that contain the proteins in an biologically active shape and form. New sur-

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faces will lead to more native “living protein arrays”. The basics for such a technology has been described by the analysis of protein-protein interactions in S. cerevisiae using the yeast two-hybrid screens in array format [11, 31]. So called “living arrays” were constructed consisting of a nearly complete set of yeast open reading frames cloned as fusions with the Gal4 activation domain. This clone set was co-transformed with a set of putative interaction partners cloned as fusions to the Gal4 DNA binding domain and subsequently arrayed on filter membranes. Protein-protein interaction was detected by arraying of the co-transformed clone set on selective media. By screening 5,345 yeast open reading frame-Gal4 activation domain fusions with 195 Gal4 DNA binding domain fusions, 957 putative interactions, involving 1,004 yeast proteins, were identified. The recombinant expression of all open reading frames of Saccharomyces cerevisiae has already been achieved. A nearly complete collection of yeast strains for the expression of 6,144 open reading frames as fusion proteins was generated, divided in pools and screened for biological activities. Collections like this can form the basis of future protein microarrays by representing a large portion of gene products. As an example for an functional assay, 119 protein kinases were expressed, purified as GST fusion proteins, arrayed and cross-linked in a protein chip format and assayed for autophosphorylation by treatment with radiolabelled ATP. Also, substrate specificity was assayed with protein chips each carrying one of a set of kinase substrates. The kinases and the radiolabelled ATP were arrayed by pipetting onto the substrate coated surfaces and phosphorylation was monitored [40–42]. In addition to all array applications, clones and their recombinant proteins will form the basis for “structural genomics”, a research field that aims to resolve the molecular structure of biomolecules and biomolecule complexes. Within the Berlin Protein Structure factory the same proteins that are deposited onto arrays are recombinantly expressed in yeast and E. coli and are subjects for crystallisation and the subsequent X-ray scattering as well as for NMR experiments. Similar technology to the technology described here is used with the immortal protein resources to do the probe preparation for structural genomics. One example is a high-precision sub-microlitre liquid dispensing system that has been developed for the preparation of hanging drop arrays for protein crystallisation. These arrays consist of 2 mL to 100 nL drops and are used to screen for suitable buffer and salt conditions for protein crystallisation [25]. The technology developed for systematic data generation in genomics and proteomics will enable us to crystallise proteins that are available only in very tiny amounts and, therefore, contribute to our understanding of genome structure and physiological function.

8 References 1. Maier E, Meier-Ewert S, Bancroft D, et al (1997) Automated array technologies for gene expression profiling. Drug Discovery Today 2(8):315–324 2. Lander ES (1999) Array of hope. Nat Genet 21(1 Suppl):3–4 3. Meier-Ewert S, Lange J, Gerst H, et al (1998) Comparative gene expression profiling by oligonucleotide fingerprinting. Nucleic Acids Res 26(9):2216–2223 4. Schena M, Shalon D, Heller R, et al (1996) Parallel human genome analysis: microarraybased expression monitoring of 1000 genes. Proc Natl Acad Sci USA 93(20):10614–10619

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Received: June 2001

CHAPTER 7

Microarray Data Representation, Annotation and Storage Alvis Brazma 1 · Ugis Sarkans 1 · Alan Robinson 1 · Jaak Vilo 1 · Martin Vingron 2 · Jörg Hoheisel 3 · Kurt Fellenberg 3 1

EMBL Outstation – Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. E-mail: [email protected] 2 Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany 3 Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, 69120 Heidelberg, Germany

Management and analysis of the huge amounts of data produced by microarray experiments is becoming one of the major bottlenecks in the utilization of this high-throughput technology. We describe the basic design of a microarray gene expression database to help microarray users and their informatics teams to set up their information services.We describe two data models – a simpler one called ArrayExpressB and the complete model ArrayExpressC, and discuss some implementation issues. For latest developments see http: www.ebi.ac.uk/arrayexpress. Keywords: Microarrays, Bioinformatics, Databases, Gene expression, Object modeling, UML

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ImageAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . ExpressionValueSet, ExpressionValue and ExpressionValueType Common Constructs . . . . . . . . . . . . . . . . . . . . . . . Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Property . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PropertyType . . . . . . . . . . . . . . . . . . . . . . . . . . . ExternalReference . . . . . . . . . . . . . . . . . . . . . . . . LiteratureReference . . . . . . . . . . . . . . . . . . . . . . . . DatabaseReference . . . . . . . . . . . . . . . . . . . . . . . . OntologyReference . . . . . . . . . . . . . . . . . . . . . . . . WebReference . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Experiment as a Whole . . . . . . . Hybridization . . . . . . . . . . . . Describing Arrays and Array Types Array and ArrayBatch . . . . . . . Element . . . . . . . . . . . . . . . Reporter . . . . . . . . . . . . . . . Extract Preparation . . . . . . . . . Biomaterial . . . . . . . . . . . . . Event . . . . . . . . . . . . . . . . Sample . . . . . . . . . . . . . . . . Array Scanning and Analysis . . . . ImageAnalysis . . . . . . . . . . . ExpressionValueSet . . . . . . . . . ExpressionValue . . . . . . . . . . ExpressionValueDimension . . . . ExpressionValueType . . . . . . . . CompositeElement . . . . . . . . . CompositeSample . . . . . . . . . . Transformation . . . . . . . . . . . Common Constructs . . . . . . . .

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1 Introduction Management and analysis of the huge amounts of data produced by microarray experiments is becoming one of the major bottlenecks in the utilization of this high-throughput technology. Organizing and storing microarray data so that they can be queried and analyzed is not a trivial problem for several reasons. First of all microarray data have meaning only in the context of the particular biological sample and exact experimental conditions under which the experiments have been performed. Developing ways to describe and represent biological conditions in a precise way that can be queried is one of the major research areas in bioinformatics. The second major problem originates from the lack of standards in microarray technologies and data analysis methods. The raw data produced by array technologies are images, while, conceptually, gene expression data is a table representing expression levels of particular genes in particular samples (for instance we could assume that each row in the table represents a gene, and each column a sample – we call such a table a gene expression matrix, while the values in the matrix are often referred to as the gene expression levels). Ideally, it would be desirable to measure the expression levels in some natural units, such as the number of mRNA molecules per cell, and also to have an error-estimate associated with each value. Unfortunately, the accuracy of most measurement procedures used at present is not sufficient to reliably assess the abundance of gene products in such objective units. Moreover, the relationship between the raw images and the gene expression matrix is not straightforward. To obtain any meaningful gene expression data from such images, they have to be quantitized and data from several images have to be combined and normalized, and the results recorded in the database. In the absence of standard controls, the gene expression data from different sources effectively use different measurement units; the relative values of which are typically unknown and may even vary depending on the measurement range. This makes it necessary to store in the database not only the gene expression data matrix, but also the raw and intermediate data, and a detailed description as to how the processed values are obtained. This complicates the database design enormously. The size of the datasets (that can easily reach terabytes) adds to the difficulties. In this paper we describe the basic design of a microarray gene expression database to help microarray users and their informatics teams set up their information services. The first version of the microarray database object model ArrayExpress, which is described here, was developed at the European Bioinformatics Institute (EBI) in collaboration with the German Cancer Research Centre (DKFZ) and posted on the Internet in November, 1999 (see (http://www.ebi.ac.uk/arrayexpress)). A relational database implementation maxd based on the ArrayExpress object model was conducted at the University of Manchester (http://bioinf.man.ac.uk/microarray/resources.html) and is widely used for microarray laboratory informatics support.

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The first ArrayExpress version was limited in several respects. Firstly, it was essentially array element (spot) centric, rather than gene centric. Concretely, the model assumed that each gene is represented by one spot on the array. It also did not allow information from replicate experiments to be easily summarized. To deal with these and other limitations we have developed a revised version of ArrayExpress, which we describe here. In fact we will describe two models – a simpler one called the Basic model (ArrayExpressB), and a more elaborate one, called the Complete model (ArrayExpressC). The new ArrayExpress model has been considerably influenced by the object model used for the Genomics Knowledge Platform developed by Incyte Genomics. A standard way to describe biological samples and conditions is needed if the expression data are to be queried and compared. This requires the use of standard ontologies and controlled vocabularies for biological sample description, which at the moment are only under development. The Microarray Gene Expression Database (MGED) group was founded in 1999 to facilitate the adoption of standards for DNA-array experiment annotation and data representation, as well as the introduction of standard experimental controls and data normalization methods. The MGED group has recently completed their first set of recommendations (see (http://www.mged.org)).At the basis of these recommendations is the formulation of the minimum information about a microarray experiment that researchers are encouraged to report in order to ensure the interpretability of the experimental results, as well as their potential verification by third parties. The recommendations are briefly described in the following sections. The database design described here can be used to capture information complying with these MGED recommendations.

2 Minimum Information About a Microarray Experiment It is important that all the information that may be needed to interpret the results of a microarray experiment at a later date, and possibly to verify the experiment, is captured and recorded in the database. The microarray data annotations working-group of MGED has developed a list of requirements that have to be satisfied to meet these goals, called the “Minimum Information About a Microarray Experiment” (MIAME). There are two simple principles underpinning MIAME. First, microarray data should be annotated in sufficient detail to be of most use to third parties. Second, while the microarray technology is still rapidly developing, it would be counterproductive to try to impose on researchers the use of any particular platforms or software, and any particular methods of data analysis. Instead, the standards should simply require revealing of data in sufficient detail. The minimum information about a published microarray based gene expression experiment includes the following six parts: 1. experimental design: the set of the hybridization experiments as a whole; 2. array design: each array used and each element (spot) on the array; 3. samples: samples used, the extract preparation and labeling;

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4. hybridizations: procedures and parameters; 5. measurements: images, quantitation, specifications; and 6. controls: types, values, specifications; Details of 1–6 can be found on (http://www.mged.org). MIAME is aimed at cooperative data providers, and not to close all possible loopholes in providing the crucial information. The goal is to produce a workable standard that neither imposes an undue burden on scientists nor limits their ability to communicate their unique results.Among the concepts in the standard are sets of ‘qualifier, value, source’ triplets, where ‘source’ is either user defined, or a reference to an externally defined ontology or controlled vocabulary, such as the species taxonomy database at NCBI (http://www.ncbi.nlm.nih.gov/Taxonomy). Where necessary, authors are encouraged to define their own qualifiers and provide the appropriate values so that the list as a whole gives sufficient information to interpret the particular part of the experiment. Judgment regarding the necessary level of detail is left to the data providers. In future, these ‘voluntary’ qualifier lists may be gradually substituted by required fields, as the respective ontologies are developed. Parts of the MIAME can be provided as a reference or link to an externally existing description. For instance, for commercial or other standard arrays, all the required information should be normally described only once by the array provider and referenced by the users. Standard protocols should also normally be stored only once. It is necessary that either a valid reference or the information itself is provided. We recommend that all the information about microarray experiments specified in MIAME is captured by the users in a database. The database design described below can be used to satisfy the MIAME requirements.

3 Structure and Design of the ArrayExpress Database The presented microarray database design allows the capture of information about all the experimental steps that are performed to measure gene expression: preparing the samples, extracting and labeling gene transcripts from each sample, hybridization of these extracts onto the array, scanning the hybridized array, analyzing the scanned image to identify and quantify each element on the array, and deriving the gene expression levels from this quantification. The last step may combine values from several elements related to the same gene (possibly from different hybridizations). The basic data unit that can be stored in the database is called Experiment.An Experiment consists of one or more Hybridizations between labeled extracts obtained from Samples and reporters that are printed on the Array. The hybridized array is analyzed by applying scanning and image analysis protocols, and an ExpressionValue is attributed to each spot on the array. The overall structure of the database, its top-level objects and the relationships between them are given in Fig. 1. One of the considerations behind the proposed design is that the model should be general enough to capture information from all existing microarray plat-

Fig. 1. Overall structure of ArrayExpress

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forms, and even some possible future platforms. For laboratories using only one particular array platform, such a generality may not be necessary, but, on the other hand, it would enable them to switch to new platforms more easily. The second aspect of the ArrayExpress model, which distinguishes it from similar simpler designs, is the ability to capture raw and intermediate data originating from microarray experiments. For instance, many of the existing public datasets report only the background-subtracted signal ratios for the popular two channel (Cy3/Cy5) technologies [1], which result in the loss of all the quality information characterizing each individual data-point. ArrayExpress allows the capture of complete outputs from image quantitation software regardless of the particular image analysis algorithm or software package used. Quantities such as the standard deviation of pixel intensities within a spot or number of pixels in a spot provide useful information about the quality of the particular data-point, which is important in many applications. Laboratories using a particular scanner and image analysis software may simplify the design, but the tradeoff between the simplicity and the flexibility to update the technology without changing the database has to be kept in mind. It should also be noted that we cannot assume that an array element (spot) and a gene have a one-to-one relationship between them – in fact, for oligonucleotide arrays, there may be far too many relationships between genes and elements on the array (for instance, if there is a need to distinguish between closely homologous genes). A one gene, many spots relationship arises also when dealing with replicate experiments. The ArrayExpress model allows the storage of the initial array element quantitations along with all the derived values which can be obtained by combining several primary element quantitations. The final derivations would normally reflect gene expression levels, either absolute or relative, depending on the particular technology. Because of these complexities, we will start by describing a simplified ArrayExpress model – ArrayExpressB, after which we will add the details of the ArrayExpressC model. Some users may not need to implement the ArrayExpressC model for their particular purposes. We encourage users to assess whether the ArrayExpressB model is sufficient or not (data structured according to the ArrayExpressB model can be later imported into the ArrayExpressC model compliant database). Also, a whole range of “intermediate” models between the two extremes is possible. In fact, these models should be critically assessed by users and adjusted to the particular needs of their laboratories. The ArrayExpressB and ArrayExpressC models are presented on Figs. 2 and 3, respectively. Models are described as Unified Modeling Language (UML) diagrams; however, so-called Object Modeling Technique (OMT) notation is used to represent association cardinalities, giving more compact representation. In the following sections we will describe the most essential feature of the ArrayExpress model, the full documentation is available on (http://www.ebi.ac.uk/arrayexpress/).

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4 Object Modeling, UML and OMT Notations The database has been designed using object-modeling techniques. In this section we give a brief introduction to the object modeling approach and explain the used notation. For a full introduction to object modeling, see [2]. An object class represents a set of real-world entities with similar properties and behavior (e.g., a DNA array) or domain abstraction of such entities (a DNA array type). Boxes in diagrams represent object classes, with the class name written above the horizontal line and attributes listed below the line. Classes have attributes. An attribute is used to describe some property common to all objects of the corresponding class (where property values can be and usually are different). An attribute can have a type – a set of potentially allowed values for this attribute. On diagrams, attributes are shown in the form ·attribute nameÒ:·attribute typeÒ. A relationship between classes represents some form of connection between objects of these classes. On diagrams, relationships are represented by lines. There are many types of relationships, three of which are used in ArrayExpress models. A class is in a subclass relationship with another class if all objects of the subclass belong also to the superclass. A subclass relationship is sometimes called “IS A” relationship (e.g.,“car IS A vehicle”). On diagrams it is represented by a triangle with its upper corner connected to the superclass and its base connected to all its subclasses. A class is in an aggregate relationship (or aggregation) with another class if objects of this class are physically and/or logically parts of that other class and cannot exist independently. This relationship is sometimes called “HAS A” relationship (e.g., car HAS A registration number). On diagrams it is represented by a line connecting the two classes, with a small diamond at the end of the container (parent) class. A class is in association with another class if there can be some other type of connection between objects of these classes than subclass or aggregation.An association can have a name, and there can be role names attached to each end of the association to characterize how an object at the corresponding end might be called if it participates in such association with an object from the other class (useful for cases when there are several associations between the same pair of classes, an association is recursive, i.e., between objects of the same class, or it is not obvious what kind of association this is). On diagrams, associations are represented as lines.An association name can be written near the middle of the line, and role names can be written near the corresponding ends of the line. On diagrams created with Rational Rose role names might be prefixed by + or some other symbol; these symbols can be ignored for purposes of conceptual modeling. Associations and aggregations can have cardinalities, indicating how many objects of one class can be associated with the same object of the other class. Each relationship may have two cardinalities, one for each end. Typical cardinalities are 1 (each object at the other end must have exactly one object at this end associ-

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ated through this relationship), 0…1 (… can have no more than one …), 0…* (.… can have any number …), 1…* (… must have at least one …). On diagrams, cardinalities are written near the corresponding relationship end. In OMT notation, which is more compact, relationship ends are transformed. No transformation means cardinality 1, small hollow circle means 0…1, small filled circle means 0…*, and small filled circle with “1+” written nearby means 1…*.

5 Basic Model – ArrayExpressB In this section we describe the most essential elements of the ArrayExpress model, particularly the ones that are important for laboratory databases. The Complete ArrayExpress model will be described in the next section. 5.1 Experiment as a Whole 5.1.1 Experiment

This is the top-level class of ArrayExpress.An object of this class represents a description of a microarray experiment as a unit. Data is entered into the database by filling in one or more Experiment class objects together with all related objects. An Experiment consists of one or more Hybridizations. Experiment class attributes are used to describe properties of a microarray experiment as a whole. They include: – Experiment type, e.g., toxicity, drug efficacy, differential expression, normal vs diseased comparison, treated vs untreated comparison, time course, dose response, effect of gene knock-out, effect of gene knock-in, shock. Multiple types are possible. – Experimental factors, e.g., time, dosage, severity, location, genetic background. – A flag indicating whether Hybridizations of this Experiment are ordered or not; actual order of Hybridizations is determined by orderNumber attribute of the Hybridization class. Experiment class has an attribute description which can hold a reference to a Description class object (described below), specifying publications in which this Experiment is documented, online supplementary resources and other relevant information which does not fit into provided attributes. 5.2 Hybridization

An object of the Hybridization class represents an instance of array hybridization, where a labeled extract from the target sample is hybridized with an array of a given array type, under a given set of conditions. A Hybridization is a part

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of an Experiment. One or more LabeledExtracts are used for a Hybridization, typically using differently labeled extracts. Hybridization results are scanned, resulting in one or more Images. A Hybridization class attribute orderNumber can be used to record ordering of Hybridizations within an Experiment (if the order is relevant). The protocol used to hybridize the LabeledExtract to the array should be described using protocol attribute. Information that can be recorded here includes: solution characteristics (e.g., concentration of solutes), blocking agent, wash procedure, quantity of labeled target used, time, volume, temperature, and description of the hybridization instruments. 5.3 Describing Arrays and Array Types 5.3.1 ArrayType

ArrayType class objects correspond to microarray designs, not individual arrays. We expect that for standard arrays this information will be provided by the array producers, either commercial or resource centers. The same ArrayType can be used for many Hybridizations; therefore, a single ArrayType object can be associated with multiple Hybridization objects. Attributes of the ArrayType class include the following attributes: – Array design name (e.g.,“Stanford Human 10K set”). – Platform type, e.g., spotted or synthesized; cDNA, oligos or PCR products; plasmids or colonies. – Array size, the number of rows and columns in the grid of Elements, or the number of elements if the layout is more complex than orthogonal rows and columns. – Array substrate: absorptive or nonabsorptive, type name (e.g., glass, nylon). – Production protocol. Laboratories making their own arrays should normally capture this information by their laboratory information management systems (LIMS), which should ideally upload this information directly into the database. 5.3.2 Element

An Element represents a single element (spot) on the array or, more precisely, design of a single element as a part of the design of related ArrayType. Elements contain reporters used to detect the target molecules. A reporter is a set of identical (in principle) DNA molecules. For an Element its row and column (or, if layout is more complex – its position) can be described using Element class attributes. Other relevant information, e.g., production protocol or size, can be captured by the description attribute. Each Element object is related to one or more Biosequence objects.

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5.3.3 Biosequence

Biosequence objects characterize the reporter contained within the respective Element. This information – what is the sequence of Element’s reporter, what gene this reporter is thought to represent – can be regarded as an attribute of Element class. However, more complex information than just a sequence string or gene name might be required (e.g., reference to sequence information in some external database), therefore Biosequence is defined as a separate class. A Biosequence object may represent actual complete sequence of the related Elements, actual partial sequence (several partial sequences can characterize a single Element), actual oligo sequence (several oligos can characterize a single Element), or a reference sequence (a sequence which is thought to represent the actual sequence(s) in the array element, e.g., if actual sequences are not known). A Biosequence object can also store clone or gene information if the related Element is described in terms of clones and/or genes, e.g., a pointer to the clone in a clone library database (if the probe has been obtained from a clone). If the sequence description is present in a biosequence database such as GenBank or EMBL bank, then it can be described by its accession number and an unambiguous database name. For ESTs the links to the known EST clusters can be given. These cross-references will also provide the links into other databases, e.g., metabolic and functional databases that describe the gene. A Biosequence can be related to several array Elements. The following information can be recorded using Biosequence class attributes: – Type of the Biosequence object, e.g., complete sequence, partial sequence, oligo sequence, reference sequence, clone, gene. – Actual sequence string, particularly if the sequence is not referenced within some database (through description → Properties → ExternalReference). – Approximate length of the Biosequence if sequence is not known precisely. 5.4 Extract Preparation 5.4.1 SampleSource

Objects of this class represent organisms, cell lines, tissues or other sample sources used in the hybridization. Descriptions of SampleSources should include organism (NCBI taxonomy) and cell source and type [if derived from primary source(s)]. An additional property list may include: sex, age, development stage, organism part (tissue), animal/plant strain or line (if applicable), genetic variation (e.g., gene knock-out, transgenic variation), individual (if applicable), individual genetic characteristics (e.g., disease alleles, polymorphisms), disease state (or indication “normal”). A standard controlled vocabulary should ideally be used to characterize the SampleSource, but since there is no generally accepted nomenclature covering all cases, this may be only partly achievable.

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One or more SampleSource objects can serve as sources for some Treatment, resulting either in another SampleSource (or SampleSources), or Sample(s), or combination of both. 5.4.2 Sample

This object describes the sample properties that can change during the experiment. One or more Sample objects can serve as sources for some Treatment, resulting in another Sample (or Samples). In addition, SamplesTreatments can also have SampleSources as treatment sources. Although initial ExpressionValues (see below) are calculated for each particular Image, the information we are really interested in is how each gene is expressed in each Sample. In ArrayExpressB we can trace ExpressionValue – Sample relations through the chain of classes ExpressionValue → ExpressionValueSet → ImageAnalysis → Image → Hybridization → LabeledExtract → Labeling → Extract → Extraction → Sample. Whether a property should be recorded in the Sample or SampleSource class depends on the particular experiment. For example, in an experiment where gene expression changes during a progression of a cancer are studied, the description of the disease stage naturally belongs to Sample class (since it changes). In a drug-response type of study, where a particular same developmental stage is used, it belongs to SampleSource. 5.4.3 Treatment

Objects of this class can be used to describe in vivo treatments (then one or more SampleSource objects are sources for this treatment and one or more SampleSource objects can be results of this treatment) or in vitro treatments, when source and/or result can be Sample object(s). Examples of such Treatments are “perform heat shock” (e.g., for yeast),“add compound”, etc. A Treatment might be simply a delay, i.e.,“wait 1 hour”. The Treatment attributes include: treatment type (e.g., heat shock, add compound), treatment compound, sample separation technique, Treatment protocol (free-text description). 5.4.4 Extraction and Extract

Objects of this class represent procedures for the isolation of the target molecules from a sample for their subsequent labeling and hybridization on an array.An Extraction uses one or more Samples and results in a single Extract. Description of how the Extract was produced from the Sample and prepared for Hybridization may include: cell rupture method, chemical extraction procedure (e.g., phenol, guanidine isothiocyanate), physical extraction procedure (e.g., centrifugation,

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poly-A beads, columns), whether total RNA, mRNA, or genomic DNA was extracted, amount of nucleic acids extracted, target amplification (RNA polymerases, PCR). 5.4.5 Labeling and LabeledExtract

Objects of the Labeling class represent instances of processes used to label Extracts, obtaining LabeledExtracts. Each Labeling uses exactly one Extract and produces exactly one LabeledExtract. Description of labeling protocol can include: label used (e.g., Cy3, Cy5, 33P), amount of nucleic acids labeled, the labeling ratio (efficiency). A LabeledExtract object is the substance that is hybridized to an array. Information on the process by which the LabeledExtract has been obtained can be traced back using relations LabeledExtract → Labeling → Extract → Extraction → Sample → Treatment → SampleSource. 5.5 Array Scanning and Analysis 5.5.1 Image

The next experimental steps after the hybridization typically are scanning of the hybridized array, which results in a digitized image (represented by Image class), followed by quantitation of the image (represented by ImageAnalysis class described below). The digitized images typically are not stored in the database, but they may be kept in a separate image archive (indexed in the database). If the image is not stored in the database, then the object of this class is a placeholder for annotation about the image and how it was produced. Some of Image attributes are: image URL – reference to the physical location of the image; protocol – used to record details of the scanning process (hardware, software, parameters, procedure); description that can include parsed header of the TIFF file, including laser power, spatial resolution, pixel space, PMT voltage. 5.5.2 ImageAnalysis

Objects of this class correspond to instances of scanned microarray image analysis. A single Image can be analyzed several times using different protocols, and several Images (from the same Hybridization) can be used for single analysis. Result of an ImageAnalysis is an ExpressionValueSet. For description of the analysis characteristics, protocol attribute should be used. The software and parameters used are specified, as well the quality assessment of the overall image (if available) and any normalization that has been applied before the final output.

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5.5.3 ExpressionValueSet, ExpressionValue and ExpressionValueType

A single ExpressionValueSet can be obtained from ImageAnalysis. An ExpressionValueSet contains many ExpressionValues. Each ExpressionValue may be linked to one or more Elements. There is an indirect link to Sample through ExpressionValueSet → ImageAnalysis → Image → Hybridization → LabeledExtract → Labeling → Extract → Extraction → Sample. Although the numerical measurement data depends on the particular image analysis algorithm, usually we can distinguish two types of ExpressionValues. The first type includes raw data that is a direct output by the image analysis software (for each spot, mean intensity, mean background, median intensity, median background, number of pixels in the spot, standard deviation of pixel intensities, etc., depending on the particular software). The second type is consolidated values that can be directly used to characterize the expression level of a particular gene (or reliability of a particular expression measurement), such as background-subtracted mean intensity for a spot. The raw information can be useful in assessing the reliability of the final expression level measurements. For example, the correlation between the intensities of the pixels in a spot is a possible reliability indicator. Ideally, we would like to be able to deduce the measurement error level from these numbers; unfortunately, currently, there are no standard reliability measures accepted (this would require an agreement on the definition of the standard error based on the analysis of only one spot). We envisage that, in the future, methods and standards for the measurement of quality assessment will be developed. In this case it may be sufficient to store only the measurement value and the quality assessment for each gene. The attributes of the ExpressionValue class are: – actual numerical value; units and other relevant information are given in the related ExpressionValueType object; – quality score of the ExpressionValue (if applicable for the particular ExpressionValueType). Categories of ExpressionValues are objects of expression value type class. Attributes include: – name of the ExpressionValueType object (e.g., “channel1 intensity”, “background pixel count”), – measurement units, – type of the qualityScore attribute of associated ExpressionValue objects (if applicable), – measurement units for qualityScores. 5.6 Common Constructs

Pieces of information, such as protocols and external references, are common for all areas of the expression data domain. For example, protocols are used to describe ImageAnalysis, as well as Hybridizations. Although these are rather dif-

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ferent kinds of protocols in terms of actions they describe, from the viewpoint of the information structure they are rather similar, therefore a common Protocol class is introduced. 5.6.1 Description

This is a catchall class to store annotation recorded in a free-text description as well as in a controlled manner using name-value pair lists for Properties. 5.6.2 Protocol

Objects of this class are referenced from protocol attribute of classes Treatment, Extraction, Labeling, Hybridization, Image, ImageAnalysis, ArrayType, ExpressionValueType. Protocol is a subclass of Description, i.e., it can consist of freetext description and properties. In the future more structured description of Protocols can be envisaged (involving, e.g., protocol steps). 5.6.3 Property

The name-value pair, e.g., age=29 years; sex=male; development_stage=adult. Value is stored in attribute value, while property name can be found in the related PropertyType object, where, if necessary, a unit for the value may also be specified. Properties are grouped to form Descriptions. A Property can have an ExternalReference attached, either documenting Property value or acting as an external Property value (then attribute value should be empty). 5.6.4 PropertyType

Objects of this class determine what types of properties are available in descriptions of objects of various ArrayExpress classes. Attributes of this class include: property name, measurement units, free-text description, list of class names determining which objects can have properties of this type. 5.6.5 ExternalReference

Any object that is an ExternalReference (i.e., LiteratureReference, DatabaseReference, OntologyReference and WebReference) can be associated with a Property that is a part of some Description. Such ExternalReference either documents how the value attribute of the Property object was obtained, or essentially provides the value (then Property → value should be empty).

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5.6.6 LiteratureReference

These can be references to publications that can be attached to any Description (since LiteratureReference is a subclass of ExternalReference). The only attribute is pubMedID; for LiteratureReferences that are not in PubMed, description attribute (of the superclass ExternalReference) should be used. Typically, LiteratureReferences will be used in Descriptions of Experiments and ArrayTypes. 5.6.7 DatabaseReference

This class is a subclass of ExternalReference class. An object of the DatabaseReference class represents either some external database as a whole, or a particular entry in that database. The most typical use of DatabaseReferences within ArrayExpress will be linking Biosequence objects to corresponding database entries in external databases. A DatabaseReference object contains a unique identifier of the database, database URL, and an identifier that permits unique identification of the relevant object within the database. 5.6.8 OntologyReference

In order to describe ArrayExpress objects (especially SampleSources and Samples), Description type objects will be used, incorporating both free text and controlled text through property name – property value pairs. It is advisable to use a reference to an external ontology where appropriate. Objects of the class OntologyReference link together property values with external ontologies. An OntologyReference object contains ontology URL (it is assumed that an external ontology has to be encoded in some XML-compliant format and should have a URL) and a reference to a specific element of the ontology. 5.6.9 WebReference

An object of this class can be used as a general way to reference some Web resource (which is not a database, ontology or article).

6 Complete Model – ArrayExpressC There are experimental setups for which the Basic model is not sufficiently detailed. For instance, ArrayExpressB does not allow the capture of ExpressionValue when it is related to more than one array Element. Also there is no way to capture relations between several ExpressionValueSets. For instance, an ExpressionValueSet can be obtained by averaging several ExpressionValueSets each obtained from different replicate Hybridizations.ArrayExpressC provides means to

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deal with these cases in a technology and expression data analysis independent way. There are also several other differences between ArrayExpressB and ArrayExpressC that are described below. 6.1 Experiment as a Whole

An Experiment can be related to one or more other Experiments through the recursive association relates.A ControlElement in this model is used to capture the situation when there is an Element on the Array that for a particular Hybridization is expected to yield some specific expression value (of type ExpressionValueType). 6.2 Hybridization

The Array class is used to represent a particular instance of an array used for hybridization, as opposed to ArrayType. This allows the description of an experiment where several Hybridizations can be performed on the same Array (i.e., instance of ArrayType). Recursive replicate relation can relate several Hybridizations regarded as replicates of the same conditions. Hybridization class is a subclass of a more general Event class (described below). 6.3 Describing Arrays and Array Types 6.3.1 Array and ArrayBatch

An Array belongs to an ArrayBatch.All Arrays derived from a certain ArrayType are physically identical, but each has a unique array ID (e.g., a barcode as used in the laboratory for tracking purposes) that differentiates it from the others. In the ArrayExpressB model, Hybridization is directly associated with ArrayType, assuming that it is not important whether several Hybridizations are performed on the same instance of some ArrayType or on several instances. Arrays of the same ArrayType can perform differently depending on which ArrayBatch they belong to. An ArrayBatch object represents a set of Arrays belonging to the same ArrayType printed/synthesized using the same raw materials. 6.3.2 Element

Each Element is associated with exactly one Reporter. Element class is a subclass of ExpressionValueDimension class. If an ExpressionValue object is related to exactly one Element, then it will be associated with it through the ExpressionValue → ExpressionValueDimension association.

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Elements on arrays are sometimes grouped within ElementBlocks, contiguous groups of Elements in terms of array layout, that in turn can be grouped to form larger ElementBlocks. ElementGroup objects represent groups of Elements that share the same type and generation protocol. Elements within a group can differ only in position and Reporters that the Elements represent. ElementGroup attributes include: element type, e.g., synthesized oligo-nucleotides, PCR products, plasmids, colonies, whether single or double stranded, expected dimensions of all elements within the group, according to Element generation protocol, reference to Protocol used for synthesizing Elements within the group, attachment type, e.g., covalent, ionic, hydrophobic, other. 6.3.3 Reporter

Reporter class objects represent sequences that are attached to arrays as Elements. Each Reporter object can have several related Element objects (either on the same ArrayType or on several ArrayTypes). Each Reporter is described by one or more (e.g., in case of several known partial sequences) Biosequence objects. Reporter class is introduced to explicitly model distinction between physical Elements on arrays and “logical” elements that are characterized by one or more Biosequence class objects. Note that there is no standard terminology for these notions.What we call Reporters are sometimes called probes or targets, in which case LabeledExtracts in our model are called targets or probes, respectively. To avoid possible confusion resulting from the opposite use of terms “probe” and “target” we avoid them entirely. 6.4 Extract Preparation 6.4.1 Biomaterial

Objects of this class can represent materials used in microarray experiments in various stages of extraction: source, sample, extract, extract after labeling. Biomaterial is a superclass of SampleSource, Sample, Extract and LabeledExtract classes. Biomaterials can be transformed by Events, resulting in new Biomaterials (or new states of the same biomaterials that should be represented by new objects of Biomaterial class). Two associations link Biomaterial and Event classes, one used for linking Biomaterial(s) as source(s) for some Event, another for linking Biomaterial(s) as result(s) of some Event. There are associations connecting subclasses of Biomaterial to subclasses of Event (Treatment, Extraction, Labeling, Hybridization), forming a typical chain of actions performed during microarray experiments. Biomaterial and Event class objects as more abstract, together with abstract Biomaterial → Event associations, should be used when this typical chain is not sufficient.

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6.4.2 Event

Labeled extracts used in microarray experiments can be traced starting from extract source (organism), through sample to extract and labeled extract. The single superclass for all these objects is Biomaterial. Objects of the Biomaterial class can be thought of as nodes in an acyclic directed graph, with arcs being Events that transform sets of Biomaterials into other sets of Biomaterials. In the ArrayExpressC model, a Hybridization is a subclass of the Event class. Event objects can be composed of several other Event objects through eventComposition recursive aggregation. Therefore, complex treatments can be described in terms of simpler treatments, and it is not necessary for each simple Event object to introduce a new Biomaterial class object representing a biomaterial state after that simple Event and before other successive Events. Each Event has its name, which ideally should characterize the treatment (e.g., heat shock, cold shock, food deprivation, add chemical, dry, centrifuge, resuspend, wait). Since Event class objects can document many types of biological manipulations, protocol for an Event can include various types of descriptions (see also descriptions of Event subclasses): – How the source was perturbed and treated in order to produce the sample (e.g., apply heat shock). It is possible to subcategorize various treatments into the following groups: in vivo treatments (organism or individual treatments), in vitro treatments (cell culture conditions), and separation technique (e.g., none, trimming, microdissection, FACS). – How an extract was produced from a sample and prepared for hybridization, e.g., extraction method, whether total RNA, mRNA, or genomic DNA was extracted, exogenous sequences (spikes) added (for each spike added: type, name/clone id, quantity added), target amplification (RNA polymerases, PCR). – How the labeling was performed. – How the labeled extract was hybridized to the array. 6.4.3 Sample

In ArrayExpressC, Sample class is a subclass of ExpressionValueDimension class, thus Sample objects can be related to ExpressionValue objects. 6.5 Array Scanning and Analysis

In the ArrayExpressC model, the description of the array scanning and analysis allows for many-to-many relationships between the array elements and expression values.

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6.5.1 ImageAnalysis

The many-to-one association between ImageAnalysis and Hybridization emphasizes that several Images can be used for single ImageAnalysis only if they are obtained from the same Hybridization. ImageAnalysis is a subclass of ExpressionValueDimension; therefore, ExpressionValues can be directly related to ImageAnalysis objects. 6.5.2 ExpressionValueSet

In ArrayExpressB an association between ImageAnalysis and ExpressionValueSet was introduced. However, an ExpressionValueSet can also be a result of some Transformation (described below). ExpressionValueSet contains ExpressionValues that have links with several ExpressionValueDimension objects. There is a direct association between ExpressionValueSet and ExpressionValueDimension. A Transformation can consume one or more ExpressionValueSets and produce one ExpressionValueSet. Each ExpressionValueSet is a product of no more than one Transformation, but it can serve as a source for several Transformations. 6.5.3 ExpressionValue

In ArrayExpressC an ExpressionValue object can have links to several ExpressionValueDimension objects. ExpressionValueDimension is an abstraction of all classes that can be directly or indirectly linked to ExpressionValues. 6.5.4 ExpressionValueDimension

ExpressionValueDimension is an abstraction of all classes that can be directly or indirectly linked to ExpressionValues. Subclasses of ExpressionValueDimension are ExpressionValueType, Element, CompositeElement, Sample, CompositeSample, ImageAnalysis. Such abstraction was introduced to permit easier modeling of Transformations (described below). These dimensions are not necessarily independent; since, e.g., the related Sample object depends on the related ImageAnalysis object, some ExpressionValueTypes are meaningful only for single Samples, some only for CompositeSample (e.g., ratio), etc. Each ExpressionValue can have up to four related ExpressionValueDimension objects: – ExpressionValueType (mandatory). – Element or CompositeElement, i.e., which Element(s) the ExpressionValue is related to. – ImageAnalysis, allowing to track which Image(s) served as a source for this ExpressionValue and what protocol was used. This is an optional dimension,

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since an ExpressionValue might be obtained by some Transformation involving several primary ExpressionValueSets resulting from several ImageAnalysis objects. – Sample or CompositeSample. This dimension depends on ImageAnalysis dimension, but was added in the model to stress that, although technically ExpressionValues are obtained through ImageAnalysis, meaningful information for further analysis is which Samples these ExpressionValues characterize. There are two relationships between ExpressionValueDimension class and Transformation class. Transformations transform ExpressionValueSets yielding new ExpressionValueSets, and they can introduce new values for expression value dimensions (i.e., new ExpressionValueDimension objects). For example, each instance of the ratio transformation transforms two raw image ExpressionValueSets into a ratio ExpressionValueSet, introducing some new ExpressionValueDimension objects: ExpressionValueType “ratio” and a new CompositeSample object. For a Transformation there may be one or more sourceDimensions, and a Transformation may yield one or more resultDimensions. 6.5.5 ExpressionValueType

For an ExpressionValueType object, a description attribute can be used to describe how this ExpressionValueType relates to its “primary” ExpressionValueTypes, if it is obtained by some Transformation. 6.5.6 CompositeElement

Some of the ExpressionValues can be computed using primary data (from image scan) originating from more than one Element (e.g., if there are several Elements on an array representing the same gene, or if the same Reporter is spotted as many Elements on a single array). Then one of the ExpressionValueDimension objects associated with this ExpressionValue will actually be an object of CompositeElement class (which is a subclass of ExpressionValueDimension), and this CompositeElement will be associated with all relevant Elements. 6.5.7 CompositeSample

Some of the ExpressionValues are related to more than one Sample (the most typical example being usual ratios). Then one of the ExpressionValueDimension objects associated with such an ExpressionValue will actually be an object of CompositeSample class (which is a subclass of ExpressionValueDimension), and this CompositeSample will be associated with all relevant Samples.

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6.5.8 Transformation

Used for tracking processing of ExpressionValueSets. Each Transformation consumes one or more ExpressionValueSets and produces an ExpressionValueSet. For example, a Transformation can average several ExpressionValueSets obtained from replicate Hybridizations, or normalize an ExpressionValueSet. A Transformation can be a composition of several simpler Transformations. A Transformation can also create new ExpressionValueDimensions. If we regard an ExpressionValueSet as a (for simplicity two-dimensional) matrix, then a Transformation can create new columns and rows in this matrix (e.g., a ratio column or an average column). SourceDimensions for this Transformation will be all ExpressionValueDimensions (i.e., rows or columns) containing ExpressionValues used to compute ExpressionValues of the new dimension(s). 6.6 Common Constructs

An additional association between Description and Property classes (called references) is added to allow Property objects belonging to one Description object to reference other Description objects. Such reference might be necessary, e.g., when there are several Descriptions of scanning hardware and we want to reference from some scanning software Description which hardware is compatible with this software. A Property belongs to one Description, but it can reference other Descriptions. Thus, Description and Property class objects can encode complex structures, if needed. There is an association between Property and Submitter classes. It can be used when database objects are annotated by database users other than (object) submitters; then source association points to the Submitter of the Property. Objects of the StandardProtocol class are referenced by Protocol class objects. A StandardProtocol object can be thought of as a protocol type, while a Protocol object is a protocol application instance. StandardProtocol is just a subclass of Description; however, in the future a more structured description of StandardProtocols can be envisaged (involving, e.g., protocol steps).

7 Implementation Issues 7.1 Database Implementation

The ArrayExpressB and ArrayExpressC object models presented here should not be regarded as optimal for relational database implementation using straightforward object model to entity-relationship model mapping. For example, creating the ExpressionValue table with a single expression value in each row may not lead to efficient implementation. Instead, single values should be grouped into larger chunks and stored in the database this way. Object model should be made

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stable, whereas underlying relational database implementation can change more often. Rational rose MDL files are available from (http://www.ebi.ac.uk/arrayexpress/). ArrayExpress implementation at EBI will run on an Oracle 8i platform; however, database schema will be easily portable to other RDBMSs. The supported data import format will be MAML, a MIAME-compliant XML language 1. Images will not be stored inside the database, they will be archived on tapes or direct access media such as CD-R or DVD-R. 7.2 Data Queries, Mining and Visualization

The ultimate purpose of the database is not just to archive the data, but to make it accessible for researchers in convenient forms. This includes the necessity to implement queries combining various properties of the data and annotations. For instance, we might look for genes in the database with expression profiles similar to a given one. An object model-based query interface to ArrayExpress is being developed. The Web interfaces for predefined types of queries will be provided on top of the general query mechanism. For the database to be used for efficient data mining and interactive visualization, extensive optimization may be required, e.g., by tuning of table indexing or producing a denormalized database schema for some parts of the database.

8 Discussion One essential question is whether to store the raw image data. Currently, there are rather opposite views in the microarray community, ranging from the view that without storing the images the database would be unreliable, to the view that storing the images in any form is cost-ineffective. When designing a database for storing information originating from physical experiments, we have to decide on the desired level of processing of the raw data before they are stored. For example, in DNA sequencing, we could treat the fluorescent traces as the raw data and the sequences as the processed data. However, a sequence is a discrete object and the error rate in determining the actual sequence is low. Therefore, the advantage of storing the sequences instead of traces is obvious (although lately the need to access the trace information as well is becoming increasingly evident). In contrast to sequence data, gene expression level measurements do not represent discrete objects, but are approximate numbers (unless the precision of the measurements was such that we could determine the precise mRNA molecule count). Moreover, the noise level in gene expression studies is much higher than in sequencing. In this case, reducing the images to numeric values may result in loss of valuable information. On the other hand, this 1

See (http://sourceforge.net/projects/mged/).

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numerical representation of images is necessary for higher-level data analysis, e.g., for clustering genes according to their expression profiles. The pros and cons for storing the images or not, or intermediate options, ranging from archiving the images separately from the database (for instance, on CD-ROMs), to storing highly compressed images suited only for “eye-balling” (which do not allow their re-analysis), should be weighed up by the individual laboratories and the most appropriate decision made. To transform the raw experimental data into a gene expression matrix, many nontrivial steps are needed, of which the image analysis is only one. For example, a gene may be spotted several times on an array, using either the same or different parts of the gene sequence. The expression level measurements of different spots representing the same gene in the same hybridization experiment may be different, and sometimes even contradictory. To obtain the conceptual table of gene expression values for different conditions, these conflicts should be resolved and the information consolidated. Even if a good consolidation procedure can be found, some experimental information is lost, unless all the expression measurements regarding the same gene are kept in the database. It may be even more difficult to consolidate information from different experiments, where different names or accession numbers are used for the same gene. The last problem is not specific for a gene expression database, but is haunting bioinformatics in general. It does not seem practical to tie the solution of this general problem with the implementation of the gene expression database. Another question that has to be considered by database developers is where to make the compromise between the database complexity and flexibility. The reason why the presented ArrayExpress model is so complicated is the need to capture data from different microarray platforms in a single model. Laboratories using only one technology platform may simplify the model. On the other hand, the microarray technology is still rapidly developing, and a more generic model may have the advantage that it may not have to be changed when the technology is upgraded. The data model is able to capture detailed experiment annotation.Annotating the experiments in such detail may be regarded as an unnecessary burden by some laboratories. In the current gene expression databases, some of the samples are annotated rather briefly, for instance as ‘control’. Laboratories should consider the tradeoffs of investing resources in annotation of their experiments in more or less detail, and being able to use the obtained data at a later date. Acknowledgements. The authors wish to thank the MGED group for its contribution in developing the requirements for the database. In particular we would like to thank Paul Spellman, from University of California at Berkeley,Alex Lash at NCBI, Harry Mangalam and Jason Stewart at NCGR, Michael Bittner at NHGRI, and Frank Holstege at the University of Utrecht. We would also like to thank the Database Applications group at the EBI, and Nicole Redaschi in particular. The development of the ArrayExpress prototype is supported by Incyte. The ArrayExpress object model was influenced by Incyte’s model that was developed as a part of Incyte’s Genomic Knowledge Platform initiative, aimed towards unifying genomics-related information sources and tools by providing a single comprehensive object model and implementing necessary middleware components. The authors wish to thank Lee Grover, Frank Russo and Greg Pelz for their contribution.

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9 References 1. The Chipping Forecast (1999) Nature Genetics 21 (Special Supplement) 2. Fowler M, Scott K, Booch G (1999) UML distilled, 2nd edn.A brief guide to the standard object modeling language, The Addison-Wesley Object Technology Series Received: June 2001